Immunogenic lipopeptides comprising T-helper and cytotoxic T-lymphocyte (CTL) epitopes

ABSTRACT

The present invention provides synthetic immunogenic lipopeptide molecules comprising co-linear T-helper and CTL epitopes, and methods for their production and use in the generation of primary and secondary immune responses, and for the vaccination of animal subjects against particular CTL epitopes. More particularly, the present invention provides highly soluble lipopeptides wherein the lipid moiety is attached to the terminal side-chain group of an internal lysine or lysine analog, preferably to the terminal side-chain group of an internal diamino acid residue. Preferably the internal lysine or lysine analog is positioned between the T-helper epitope and the CTL epitope.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/524,936,filed Oct. 20, 2005,now U.S. Pat. No. 7,833,532, which is the NationalStage of International Application No. PCT/AU2003/001019, filed Aug. 12,2003, which claims the benefit of and priority to U.S. Provisional.Application No. 60/403,328, filed Aug. 12, 2002; all of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the field of immunology; andmore particularly to reagents for generating cellular responses to apeptide immunogen, and methods for using said reagents for enhancing theimmune response of a subject, or for the vaccination of a subject. Evenmore specifically, the present invention relates to novel lipopeptideshaving enhanced immunogenic activity, specifically an enhanced abilityto activate a T cell response to a CD8+ T cell epitope to induce cellmediated immunity against an invading pathogen or tumour cell. Thepresent invention also provides formulations and vaccine compositionscomprising said lipopeptides, such as, for example, in combination witha pharmaceutically acceptable carrier or excipient, and methods formaking and using the formulations and vaccine compositions of theinvention.

BACKGROUND TO THE INVENTION

1. General

This specification contains amino acid sequence information preparedusing PatentIn Version 3.1, presented herein after the Abstract. Eachsequence is identified in the sequence listing by the numeric indicator<210> followed by the sequence identifier (e.g. <210>1, <210>2, etc).The length of each sequence and source organism are indicated byinformation provided in the numeric indicator fields <211> and <213>,respectively. Sequences referred to in the specification are defined bythe term “SEQ ID NO:”, followed by the sequence identifier (eg. SEQ IDNO: 1 refers to the sequence designated as <400>1).

As used herein the term “derived from” shall be taken to indicate that aspecified integer may be obtained from a particular source albeit notnecessarily directly from that source.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated step Or element orinteger or group of steps or elements or integers but not the exclusionof any other step or element or integer or group of elements orintegers.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specificexamples described herein. Functionally-equivalent products,compositions and methods are clearly within the scope of the invention,as described herein.

All the references cited in this application are specificallyincorporated by reference herein.

The present invention is performed without undue experimentation using,unless otherwise indicated, conventional techniques of molecularbiology, microbiology, virology, recombinant DNA technology, peptidesynthesis in solution, solid phase peptide synthesis, and immunology.Such procedures are described, for example, in the following texts thatare incorporated by reference:

-   1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory    Manual, Cold Spring Harbor Laboratories, New York, Second Edition    (1989), whole of Vols I, II, and III;-   2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover,    ed., 1985), IRL Press, Oxford, whole of text;-   3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait,    ed., 1984) IRL Press, Oxford, whole of text, and particularly the    papers therein by Gait, pp 1-22; Atkinson et al., pp 35-81; Sproat    et al., pp 83-115; and Wu et al., pp 135-151;-   4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames    & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;-   5. Animal Cell Culture: Practical Approach, Third Edition    (John R. W. Masters, ed., 2000), ISBN 0199637970, whole of text;-   6. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL    Press, Oxford, whole of text;-   7. Perbal, B., A Practical Guide to Molecular Cloning (1984);-   8. Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic    Press, Inc.), whole of series;-   9. J. F. Ramalho Ortigão, “The Chemistry of Peptide Synthesis” In:    Knowledge database of Access to Virtual Laboratory website    (Interactive, Germany);-   10. Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L.    (1976). Biochem. Biophys. Res. Commun. 73 336-342-   11. Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154.-   12. Barany, G. and Merrifield, R. B. (1979) in The Peptides    (Gross, E. and Meienhofer, eds.), vol. 2, pp. 1-284, Academic Press,    New York.-   13. Wünsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls    Methoden der Organischen Chemie (Müler, E., ed.), vol. 15, 4th edn.,    Parts 1 and 2, Thieme, Stuttgart.-   14. Bodanszky, M. (1984) Principles of Peptide Synthesis,    Springer-Verlag, Heidelberg.-   15. Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide    Synthesis, Springer-Verlag, Heidelberg.-   16. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.-   17. Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir    and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications).

2. Description of the Related Art

Immunotherapy or vaccination are attractive for the prophylaxis ortherapy of a wide range of disorders, such as, for example, certaininfectious diseases, or cancers. However, the application and success ofsuch treatments are limited in part by the poor immunogenicity of thetarget CTL epitope. Synthetic peptides, representing T cell immunogenselicit only weak immunity when delivered in isolation and as aconsequence, are not effective in vaccine compositions. Full-lengthproteins containing CTL epitopes do not efficiently enter the MHC classI processing pathway. Additionally, CTL epitopes are HLA-restricted andthe large degree of HLA polymorphism in human populations means thatCTL-based vaccines may not provide broad coverage to all genotypeswithin a population.

Several techniques are used to enhance the immune response of a subjectto a peptide immunogen.

It is known that utilization of an adjuvant formulation that isextrinsic to the peptide immunogen (i.e. it is mixed with the immunogenprior to use), such as, for example, complete Freund's adjuvant (CFA),will enhance the immune response of a subject to a peptide immunogen.However, many of the adjuvants currently available are too toxic for usein humans, or simply ineffective. Moreover, adjuvants of this typerequire prior formulation with the peptide immunogen—immediately beforeadministration, such formulations often having a low solubility or beinginsoluble.

Lipopeptides, wherein a lipid moiety that is known to act as an adjuvantis covalently coupled to a peptide immunogen, may be capable ofenhancing the immunogenicity of an otherwise weakly immunogenic peptidein the absence of an extrinsic adjuvant [Jung et al., Angew Chem, Int EdEngl 10, 872, (1985); Martinon et al., J Immunol 149, 3416, (1992);Toyokuni et al., J Am Chem Soc 116, 395, (1994); Deprez, et al., J MedChem 38, 459, (1995); and Sauzet et al., Vaccine 13, 1339, (1995);BenMohamed et al., Eur. J. Immunol. 27, 1242, (1997); Wiesmuller at al.,Vaccine 7, 29, (1989); Nardin et al., Vaccine 16, 590, (1998);Benmohamed, et al. Vaccine 18, 2843, (2000); and Obert, et al., Vaccine16, 161, (1998)]. Suitable lipopeptides show none of the harmful sideeffects associated with adjuvant formulations, and both antibody andcellular responses have been observed against lipopeptides.

Several different fatty acids are known for use in lipid moieties.Exemplary fatty acids include, but are not limited to, palmitoyl,myristoyl, stearoyl and decanoyl groups or, more generally, any C₂ toC₃₀ saturated, monounsaturated, or polyunsaturated fatty acyl group isthought to be useful.

The lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteine,also known as Pam₃Cys or Pam₃Cys-OH (Wiesmuller et al., Z. Physiol.Chem. 364 (1983), p 593), is a synthetic version of the N-terminalmoiety of Braun's lipoprotein that spans the inner and outer membranesof Gram negative bacteria. Pam₃Cys has the structure of Formula (I):

U.S. Pat. No. 5,700,910 to Metzger et al (Dec. 23, 1997) describesseveral N-acyl-S-(2-hydroxyalkyl)cysteines for use as intermediates inthe preparation of lipopeptides that are used as synthetic adjuvants, Blymphocyte stimulants, macrophage stimulants, or synthetic vaccines.Metzger et al. also teach the use of such compounds as intermediates inthe synthesis of Pam₃Cys-OH (Wiesmuller et al., Z. PhysioL Chem. 364(1983), p 593), and of lipopeptides that comprise this lipoamino acid oran analog thereof at the N-terminus. The lipopeptides are prepared bycoupling a lipoamino acid moiety to the peptide moiety during thesynthesis process.

Pam₃Cys when coupled to a CTL epitope peptide has been shown to becapable of stimulating virus-specific cytotoxic T lymphocyte (CTL)responses against influenza virus-infected cells (Deres et al., Nature342, 561, 1989) and to elicit protective antibodies againstfoot-and-mouth disease (Wiesmuller et al., Vaccine 7, 29, 1989; U.S.Pat. No. 6,024,964 to Jung et al., Feb. 15, 2000) when coupled to theN-terminus of an appropriate synthetic B cell epitope.

Recently, Pam₂Cys (also known as dipalmitoyl-5-glyceryl-cysteine orS-[2,3-bis(palmitoyloxy)propyl]cysteine), an analogue of Pam₃Cys, hasbeen synthesised (Metzger, J. W., A. G. Beck-Sickinger, M. Loleit, M.Eckert, W. G. Bessler, and G. Jung. 1995. J Pept Sci 1:184.) and beenshown to correspond to the lipid moiety of MALP-2, amacrophage-activating lipopeptide isolated from mycoplasma (Sacht, G.,A. Marten, U. Deiters, R. Sussmuth, G. Jung, E. Wingender, and P. F.Muhlradt. 1998. Eur J Immunol 28:4207: Muhlradt, P. F., M. Kiess, H.Meyer, R. Sussmuth, and G. Jung. 1998. Infect Immun 66:4804: Muhlradt,P. F., M. Kiess, H. Meyer, R. Sussmuth, and G. Jung. 1997. J Exp Med185:(1951). Pam₂Cys has the structure of Formula (II):

Pam₂Cys is reported to be a more potent stimulator of splenocytes andmacrophages than Pam₃Cys (Metzger et al., J Pept. Sci 1, 184, 1995;Muhlradt et al., J Exp Med 185, 1951, 1997; and Muhlradt et al., InfectImmun 66, 4804, 1998).

Generation of a strong CD8+ T cell response against a given CTL epitoperequires the generation of a strong T helper cell response. CD4⁺T-helper cells function in cell-mediated immunity (CMI) by secretingsufficient cytokines, such as, for example IL-2, to thereby facilitatethe expansion of CD8⁺ T cells or by interacting with the antigenpresenting cell (APC) thereby rendering it more competent to activateCD8⁺ T cells. Accordingly, it is desirable to administer a CTL epitopein conjunction with at least one T-helper cell epitope (Vitiello et al.,J. Clin. Invest. 95, 341-349, 1995; Livingston et al., J. Immunol. 159,1383-1392, 1997). These epitopes are recognized by T-helper cells in thecontext of MHC class II molecules on the surface of the APC.

The CTL epitope or isolated epitope can be administered in conjunctionwith a large protein having a range of T helper epitopes in order toaccommodate the diversity of class II alleles within a population ofindividuals. Alternatively, promiscuous or permissive T-helperepitope-containing peptides are administered in conjunction with the CTLepitope or epitopes. Promiscuous or permissive T-helperepitope-containing peptides are presented in the context of a vastmajority of MHC class II haplotypes, such that they induce strong CD4⁺ Thelper responses in the majority of an outbred human population.Examples of promiscuous or permissive T-helper epitopes are tetanustoxoid peptide, Plasmodium falciparum pfg27, lactate dehydrogenase, andHIVgp120 (Contreas et al., Infect. Immun, 66, 3579-3590, 1998; Gaudeboutet al., J. A.I.D.S. Human Retrovirol 14, 91-101, 1997; Kaumaya et al.,J. Mol. Recog. 6, 81-94, 1993; and Fern and Good J. Immunol. 148,907-913, 1992). Ghosh et al., Immunol 104, 58-66, 2001 and InternationalPatent Application No. PCT/AU00/00070 (WO 00/46390) also describepromiscuous T-helper epitopes from the fusion protein of CanineDistemper Virus (CDV-F). Certain promiscuous T-helper epitopes promotestrong CTL responses to a given CTL epitope, and can bypass certainhaplotype restricted immune responses (Kaumaya et al., J. MoL Recog. 6,81-94, 1993).

Routinely, a vaccine preparation will comprise a mixture of polypeptidescomprising the T-helper cell epitope and CTL epitope, however it is alsoknown to consist of a single polypeptide comprising both the T-helperepitope and the CTL epitope.

SUMMARY OF THE INVENTION

In work leading up to the present invention, the inventors sought toimprove methods for producing highly immunogenic lipopeptides having alipid moiety and a polypeptide moiety comprising both a T helper epitopeand a CTL epitope against which an immune response is desired. Theinventors showed that a highly immunogenic lipopeptide comprising bothT-helper and CTL epitopes can be produced by synthesizing a singlepolypeptide molecule comprising said epitopes with an internal lysineresidue or internal lysine analog residue and then coupling the lipidmoiety to the side-chain amino group of said internal lysine residue orsaid internal lysine analog residue, as opposed to the N-terminalattachments described previously. This enables the lipopeptide of thepresent invention to be synthesized conveniently using a single aminoacid chain, thereby requiring no post-synthesis modification toincorporate both epitopes.

By positioning said one or more lysine residue(s) or one or moreinternal lysine analog residue(s) at predetermined locations within thepolypeptide during peptide synthesis, the attachment site of the lipidis readily specified. Positioning of the lipid moiety in the lipopeptidecan thus be targeted to enhance the utility of the end-product forvaccine or adjuvant formulations.

The inventors have found that attachment of the lipid moiety via theside-chain epsilon-amino group of an internal lysine residue or theterminal side-chain group of an internal lysine analog residuepositioned between the amino acid sequences of the T helper epitope andthe CTL epitope, provides an enhancement of dendritic cell maturationwhen compared to linear structures obtained in which lipid is attachedto the N-terminus of the peptide.

One advantage provided by the lipopeptides of the present invention isthat they are sufficiently immunogenic such that it is generally notnecessary to include an extrinsic adjuvant in vaccine formulationscomprising these lipopeptides.

The present invention clearly encompasses the attachment of a lipidmoiety via the epsilon-amino group of an internal lysine residue or theterminal side-chain group of an internal lysine analog residue presentin the amino acid sequence of the T helper epitope or the amino acidsequence of the CTL epitope, the only requirement being that the lipidmoiety is not attached to the N-terminus or the C-terminus of thepeptide. By “internal” means at a location other than the N-terminus orthe C-terminus of a polypeptide comprising a T helper epitope and CTLepitope. As will be known to the skilled person, solubility of a vaccinecontaining CTL epitopes is highly desirable for producing vaccineformulations on a commercial basis.

Preferably, the lipid moiety is attached via the epsilon-amino group ofa lysine residue or via the terminal side-chain group of a lysine analogresidue positioned between the amino acid sequences of the T helperepitope and the CTL epitope.

Optionally, one or more amino acid spacers is added between the T-helperepitope and the CTL epitope, such as, for example, at either side of aninternal lysine or lysine analog positioned between said epitopes.

A spacer of any conventional type can also be added between the lipidmoiety and the polypeptide moiety. Particularly preferred spacers inthis context consist of serine dimers, trimers, tetramers, etc.Alternative spacers, such as, for example, arginine dimers, trimers,tetramers, or 6-aminohexanoic acid, are also contemplated for use inthis context.

As exemplified herein, the present inventors produced the lipopeptide ofthe invention by coupling the lipid moiety to an exposed epsilon-aminogroup of an internal lysine residue in the synthetic peptide moiety.Optionally, a spacer may be added to the exposed epsilon amino groupbefore addition of the lipid moiety.

As will be apparent from the disclosure herein, a lipoamino acid ofFormula (III) or (IV) may be added directly to the epsilon amino groupof the internal lysine residue or to the terminal side-chain group of aninternal lysine analog residue. Lipoamino acids selected from the groupconsisting of: (i) Pam₂Cys (also known asdipalmitoyl-5-glyceryl-cysteine orS-[2,3-bis(palmitoyloxy)propyl]cysteine), (ii) Ste₂Cys (also known asdistearoyl-5-glyceryl-cysteine orS-[2,3-bis(stearoyloxy)propyl]cysteine), Lau₂Cys (also known asdilauroyl-5-glyceryl-cysteine or S-[2,3-bis(lauroyloxy)propyl]cysteine),and Oct₂Cys (also known as dioctanoyl-S-glyceryl-cysteine orS-[2,3-bis(octinoyloxy)propyl]cysteine) are also useful.

As exemplified herein, lipopeptides of the invention directed againstinfluenza virus induced a virus-specific CTL response in the absence ofexternal adjuvant, as reflected by their ability to induce potentCTL-mediated virus clearing responses, to induce CD8+ T cell migrationto the lungs and to upregulate the surface expression of MHC class IImolecules on immature dendritic cells (DC). The enhanced maturation ofdendritic cells following administration of the subject lipopeptides isconsistent with enhanced T-helper epitope presentation compared tolipopeptides having N-terminally coupled lipid.

As will be clear to those skilled in the art, the nature of the T-helperand CTL epitopes is not critical in the context of the presentinvention. The novel approach of attaching the lipid moiety to theepsilon amino group of one or more internal lysine residues or lysineanalogue residues within the polypeptide portion of the construct hasbroad application. Accordingly, based on the results presented herein,it will be understood that a wide range of T-helper and CTL epitopes canbe used in the lipopeptide constructs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation showing the generalized structureof the peptide and lipopeptide constructs used in this study. Peptidestructures consisted of a CD4⁺ helper T cell epitope [Th] and a CTL cellepitope [CTL] assembled as tandem linear sequences with a linkinginternal lysine residue (i.e. [Th]-Lys-[CTL]) or without any internallysine (i.e. [Th]-[CTL]). Lipopeptides were branched structures whereina lipid moiety was attached through the epsilon-amino group of a lysineresidue, Lys, situated between the two epitopes at the approximatecentre of the molecule (i.e. [Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL];[Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL]; or [Th]-Lys(Pam₁Cys-Ser-Ser)-[CTL]. Inthe case of branched constructs, the centrally located lysine residue towhich the lipid is attached is denoted in italics, Lys. In some casestwo serine residues (Ser-Ser) were added between the peptide and lipidmoiety. For the lipopeptide Pal₂LysLys[Th]-[CTL], two palmitic acidresidues were attached to the alpha and epsilon-amino groups of theN-terminal lysine residue and [Th] was attached to the epsilon-aminogroup of the penultimate lysine in the amino acid sequence. In the caseof [Th]-Lys(Chol₂Lys-Ser-Ser)-[CTL], two residues of cholesterol wereattached to an N-terminal lysine residue.

FIG. 2 is a representation of the primary amino acid sequences of thepeptide moieties attached to the lipid moieties for the structures shownin FIG. 1. Non-lipidated peptides comprising these amino acid sequenceswere designated as follows:

-   (i) [Th] consisting of a CD4+ T-helper epitope from the light chain    of influenza virus hemagglutinin as set forth in SEQ ID NO: 1;-   (ii) [CTL] consisting of an immunodominant H-2^(d)-restricted CTL    epitope consisting of amino acid residues 147-155 of the    nucleoprotein of influenza virus strain A/Puerto Rico/8/34 (PR8;    H1N1) as set forth in SEQ ID NO: 2;-   (iii) [Th]-[CTL] consisting of a polypeptide having (i) and (ii).    The sequence of the assembled peptide is shown in SEQ ID NO: 3;-   (iv) [Th]-Lys-[CTL] consisting of a polypeptide having (i) and (ii)    separated by a lysine residue (bold underlined residue). The    sequence of the assembled peptide is shown in SEQ ID NO: 4;-   (v) [P25]-Lys-[SIINFEKL] (SIINFEKL disclosed as SEQ ID NO: 173)    consisting of a T-helper epitope from CDV-F protein designated P25    (SEQ ID NO: 20) and a CTL epitope from ovalbumin (SEQ ID NO: 173)    separated by a lysine residue (bold underlined residue). The    sequence of the assembled peptide is shown in SEQ ID NO: 174;-   (vi) [P25]-Lys-[LLO91-99] consisting of a T-helper epitope from    CDV-F protein designated P25 (SEQ ID NO: 20) and a CTL epitope from    Listeria monocytogenes (SEQ ID NO: 172) separated by a lysine    residue (bold underlined residue). The sequence of the assembled    peptide is shown in SEQ ID NO: 175. And-   (vii) [P25]-Lys-[HCV] consisting of a T-helper epitope from CDV-F    protein designated P25 (SEQ ID NO: 20) and a CTL epitope from the    core protein of hepatitis C virus (SEQ ID NO: 176) separated by a    lysine residue (bold underlined residue). The sequence of the    assembled peptide is shown in SEQ ID NO: 177.

Lipopeptides comprising these amino acid sequences were designated asfollows:

-   (i) [Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL] consisting of peptide    [Th]-Lys-[CTL] (i.e. SEQ ID NO: 4) and a lipid of the Formula (III)    conjugated to the epsilon-amino group of the internal lysine (bold    underlined residue);-   (ii) [Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] consisting of peptide    [Th]-Lys-[CTL] (i.e. SEQ ID NO: 4) and a lipid of the Formula (IV)    conjugated to the epsilon-amino group of the internal lysine (bold    underlined residue);-   (iii) [P25]-Lys(Pam₂Cys-Ser-Ser)-[LLO91-99] consisting of peptide    [P25]-Lys-[LLO91-99] and a lipid of the Formula (IV) conjugated to    the epsilon-amino group of the internal lysine (bold underlined    residue) of said peptide;-   (iv) [P25]-Lys(Pam₂Cys-Ser-Ser)-[SIINFEKL] (SIINFEKL disclosed as    SEQ ID NO: 173) consisting of peptide [P25]-Lys-[SIINFEKL] (SIINFEKL    disclosed as SEQ ID NO: 173) and a lipid of the Formula (IV)    conjugated to the epsilon-amino group of the internal lysine (bold    underlined residue) of said peptide; and-   (v) [P25]-Lys(Pam₂Cys-Ser-Ser)-(HCV) consisting of peptide    [P25]-Lys-[HCV] and a lipid of the Formula (IV) conjugated to the    epsilon-amino group of the internal lysine (bold underlined residue)    of said peptide.

FIG. 3 is a graphical representation showing the reduced viral load ofmice primed with lipopeptides referred to in the legend to FIG. 1 andsubsequently challenged with influenza virus. Mice were inoculatedintranasally with 9 nmol of the lipopeptides[Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL] and [Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] in50 μl PBS (columns 2 and 3, respectively), or for the [Th]-Lys-[CTL]peptide in 50 μl PBS (column 1), or with PBS alone (column 4). Peptideand lipopeptide designations are as for the legend to FIG. 2. On day 9post immunization, mice were anesthetized using penthrane and challengedintranasally with 30,000 plaque forming units of influenza virus subtypeH3N1 known as A/Memphis/1/71 (Mem 71). Five days later, their lungs wereremoved and assayed for the presence of infectious virus by plaque assayon MDCK cells. Each bar represents the geometric mean titre of viraltitres from a group of 5 BALB/c mice and error bars represent thestandard deviation of the mean. Numbers above the bars represent thepercentage reduction in lung viral titre relative to the PBS control.

FIG. 4 a is a graphical representation showing enhancedlipopeptide-induced viral clearance in immunized mice receiving thelipopeptides referred to in the legend to FIG. 2. Mice were inoculatedwith 9 nmoles of the lipopeptides [Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL] and[Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] in 50 μl PBS (columns 2 and 3,respectively), or for the [Th]-Lys-[CTL] peptide in 50 μl PBS (column1), or with PBS alone (column 4). On day 28 post immunization, mice werechallenged with 30,000 plaque forming units of Mem 71 virus. Peptide andlipopeptide designations are as for the legend to FIG. 2. Data areexpressed as the percentage reduction in lung viral titre on day 5 postchallenge. Data show enhanced reduction in infectious virus in the lungsof mice immunized with the lipopeptides [Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL](column 2) or [Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] (column 3) compared topeptide alone (column 1) or PBS alone (column 4) at 5 dayspost-challenge.

FIG. 4 b is a graphical representation showing enhanced T cellactivation in immunized mice receiving the lipopeptides referred to inthe legend to FIG. 2. Mice were inoculated with 9 nmoles of thelipopeptides [Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL] and[Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] in 50 μl PBS (columns 2 and 3,respectively), or for the [Th]-Lys-[CTL] peptide in 50 μl PBS (column1), or with PBS alone (column 4) Immunized mice were killed 9 dayspost-immunization and a bronchio-alveolar lavage (BAL) performed.Adherent cells were removed by incubation of the BAL sample in a petridish at 37° C. for 1 hour. The non-adherent cells were recovered andstained for CD8 and CD4 expression. The cells were analyzed by flowcytometry. The lymphocyte population was identified based on the forwardand side scatter profile and 10,000 lymphocytes were analysed. Data areexpressed as the percentage of non-adherent cells in the BAL fluid thatare CD8+ lymphocytes. Data show enhanced activation of virus-specificCD8+ T cells in the BAL samples from mice immunized with thelipopeptides [Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL] (column 2) or[Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] (column 3) compared to peptide alone(column 1) or PBS alone (column 4) at 5 days post-challenge. Peptide andlipopeptide designations are as for the legend to FIG. 2.

FIG. 4 c is a graphical representation showing enhanced maturation ofdendritic cells in response to the lipopeptides referred to in thelegend to FIG. 2. A line of BALB/c splenic-derived dendritic cells (D1cells) were incubated overnight with 0.45 nmoles/mL of the peptide[Th]-Lys-[CTL] (column 1) or the lipopeptides lipopeptides[Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL] (column 2) or[Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] (column 3) or with medium alone as anegative control (column 4) or lipopolysaccharide as a positive control(LPS; column 5). The percentage of D1 cells expressing high levels ofsurface MHC class II molecules, and therefore in a mature state, weredetermined by flow cytometry. Peptide and lipopeptide designations areas for the legend to FIG. 2. Data show enhanced expression of MHC classII molecules on the surface of dendritic cells (ie. enhanced dendriticcell maturation) following exposure to the peptides[Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL] or [Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL]compared to peptide alone or medium alone.

FIG. 5 is a graphical representation showing the induction of pulmonaryviral clearing responses in mice inoculated with synthetic immunogensindicated on the x-axis, that each include the CD4⁺ T-helper epitope setforth in SEQ ID NO: 1 and the H-2^(d)-restricted CTL epitope set forthin SEQ ID NO: 2. Groups of 5 mice were immunised intranasally with 9nmoles of the specified lipopeptides in PBS. Mice were challenged 28days after priming with 10^(4,5) PFU of Mem71 influenza virusintranasally. Titres of infectious virus in lung homogenates sampled 5days following challenge were determined by plaque formation on MDCKcell monolayers. Each circle represents the virus titre of an individualmouse and the line represents the geometric mean titre of the group. Thepercentage reduction in mean viral titre relative to the PBS controlgroup is shown above each column of data.

FIG. 6 is a graphical representation showing accelerated influx of CTLderminant-specific CD8⁺-T cells into the lungs of lipopeptide-vaccinatedmice during virus challenge. Lipopeptides comprised the CD4⁺ T-helperepitope set forth in SEQ ID NO: 1 and the H-2^(d)-restricted CTL epitopeset forth in SEQ ID NO: 2. Groups of three mice were inoculatedintranasally with 9 nmole of the indicated lipopeptides intranasally. Onday 28 post priming, they were challenged intranasally with 10^(4.5) PFUof Mem71 influenza virus intranasally. CTL determinant-specificIFN-gamma-secreting cells were enumerated in the lungs of mice on day 5post-challenge by an intracellular cytokine production assay. 10,000CD8+ cells were analysed for each sample. Data represent the mean andstandard deviation for each group of mice.

FIG. 7 is a graphical representation showing show accelerated influx ofCTL-determinant-specific CD8 T cells into the lungs in mice inoculatedwith lipopeptides following viral challenge. Lipopeptides comprised theCD4⁺ T-helper epitope set forth in SEQ ID NO: 1 and theH-2^(d)-restricted CTL epitope set forth in SEQ ID NO: 2. Mice wereinoculated intranasally with 9 nmole of the specified lipopeptides inPBS. Nine days after inoculation mice were challenged intranasally with10^(4.5) PFU of Mem71 influenza virus. On day 5 post infection,CTL-determinant-specific CD8 T cells in the lungs were enumerated bystaining the lymphocytes from the lungs with anti-CD8 antibody and withtetrameric MHC class I complexes loaded with the CTL epitope. A total of30,000 CD8 T cells were analysed.

FIG. 8 is a graphical representation showing cytotoxic T cell activityin naïve mice. CTL determinant specific cytotoxicity in vivo wasmeasured using syngeneic spleen cells pulsed with the CTL determinantand labelled with high intensity CFSE. Non-pulsed spleen cells labelledwith low intensity CFSE were used as a control. A mixture of 15×10⁶cells of each target cell population was injected intravenously on day 4post-infection into naïve mice. The mice were killed 16 hr later andspleens were analysed for the presence of CFSE-high and CFSE-low cellpopulations by flow cytometry. A total of 1×10⁶ lymphocytes wereanalysed for each sample.

FIG. 9 is a graphical representation showing cytotoxic T cell activityin lipopeptide-primed mice. A mouse was inoculated intranasally with 9nmoles [TN-Lys(Pam₂Cys-Ser-Ser)-[CTL] comprising the CD4⁺ T-helperepitope set forth in SEQ ID NO: 1 and the H-2^(d)-restricted CTL epitopeset forth in SEQ ID NO: 2, in PBS. Mice were challenged with Mem71 onday 28. CTL determinant specific cytotoxicity in vivo was measured usingsyngeneic spleen cells pulsed with the CTL determinant and labelled withhigh intensity CFSE. Non-pulsed spleen cells labelled with low intensityCFSE were used as a control. A mixture of 15×10⁶ cells of each targetcell population was injected intravenously on day 4 post-infection intothe lipopeptide-primed and challenged mice. The mice were killed 16 hrlater and spleens were analysed for the presence of CFSE-high andCFSE-low cell populations by flow cytometry. A total of 1×10⁶lymphocytes were analysed for each sample.

FIG. 10 is a graphical representation showing the ability of variouspeptide-based immunogens to induce epitope-specific CTL. Lipopeptidescomprised the CD4⁺ T-helper epitope set forth in SEQ ID NO: 1 and theH-2^(d)-restricted CTL epitope set forth in SEQ ID NO: 2. Groups ofthree mice were inoculated intranasally with various lipopeptides in PBSand challenged with Mem71 on day 28. In order to analyze CTL determinantspecific cytotoxicity in vivo, syngeneic spleen cells were pulsed withthe CTL determinant and labelled with high intensity CFSE.Antigen-specific lysis was controlled by co-injecting syngeneic spleencells labelled with low intensity CFSE. A mixture of 15×10⁶ cells ofeach target cell population was injected intravenously on day 4post-infection. The mice were killed 16 hr later and spleens wereanalysed for the presence of CFSE-high and CFSE-low cell populations byflow cytometry. A total of 1×10⁶ lymphocytes were analysed for eachsample. Individual mice are represented by the closed squares and thebars represent the geometric mean titre.

FIG. 11 is a graphical representation showing induction ofinterferon-gamma producing cells by lipopeptide. Peptide comprising aT-helper epitope and a CTL epitope of Listeria monocytogenes linked viathe epsilon amino group of an internal lysine residue positioned betweensaid epitopes to Pam₂Cys (i.e. the peptide[P25]-Lys(Pam₂Cys-Ser-Ser)-[LLO91-991 listed in FIG. 2 and based uponSEQ ID NO: 175), or lipopeptide(s) based on this structure in whichPam₂Cys was linked through the epsilon amino group of said lysine, wereused to inoculate mice. Five BALB/c mice were inoculated intravenouslywith bacteria, or subcutaneously with either 9 nmoles of lipidatedpeptide [P25]-Lys(Pam₂Cys-Ser-Ser)-[LLO91-99] or 9 nmoles ofnon-lipidated peptide [P25]-Lys-[LL091-99] (SEQ ID NO: 175; FIG. 2) orphosphate buffered saline (PBS), as indicated on the x-axis. Splenocyteswere obtained from the immunized animals and stimulated in vitro witheither the isolated CTL epitope having the sequence set forth in SEQ IDNO: 172 (open bars) or no antigen (filled bars), and the number of(IFN-gamma) producing cells present was measured 28 days later. Theordinate indicates the number of IFN-gamma producing cells per 1,000,000splenocytes. Data show enhanced numbers of IFN-gamma producing cells formice immunized with lipopeptide, indicating an enhanced ability of thelipopeptides to activate T cells relative to non-lipidated peptide.

FIG. 12 is a graphical representation showing enhanced protectionagainst L. monocytogenes infection for mice immunized with thelipopeptide designated [P25]-Lys(Pam₂Cys-Ser-Ser)-[LL091-99] in FIG. 2(based upon SEQ ID NO: 175). Five 5 BALB/c mice were inoculatedintravenously with 1,000 bacteria (column 1), or immunizedsubcutaneously with PBS (column 2) or 9 nmol[P25]-Lys(Pam₂Cys-Ser-Ser)-[LLO91-99] peptide (column 3) or 9 nmolnon-lipidated [P25]-Lys-[LLO91-99] peptide (SEQ ID NO: 175; column 4),as indicated on the x-axis. Mice were challenged with whole bacteria andthe number of colony forming units present in liver was measured 28 dayspost-challenge (ordinate).

FIG. 13 is a graphical representation showing protection against B16melanoma with lipopeptide vaccination. C57BL/6 mice were vaccinated with20 nmoles lipidated peptide[P25]-Lys(Pam₂Cys-Ser-Ser)-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO:173) (open circles), non-lipidated peptide [P251-Lys-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO: 173) (open triangles) or with PBS(open squares) subcutaneously in the base of the tail. Mice were thenchallenged s.c. on the back 14 days later with 2×10⁵ B16-OVA cells (n=6per group) and tumour growth monitored as described (Anraku, et al., J.Virol. 76; 3791-3799, 2002).

FIG. 14 is a graphical representation showing therapeutic treatment ofLewis Lung tumour with a lipopeptide immunogen, as determined by thepercentage of animals that remain tumor free following immunization.Mice were injected with 3×10⁴ Lewis Lung tumour cells that had beentransfected with ovalbumin and therefore expressed the CTL epitopeSIINFEKL (SEQ ID NO: 173) [Nelson et al., J Immunol. 166: 5557-5566,2001]. Four days after receiving tumour cells, animals were inoculatedwith 20 nmoles lipidated peptide [P25]-Lys(Pam₂Cys-Ser-Ser)-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO: 173) (open circles), non-lipidatedpeptide [P25]-Lys-[SIINFEKL] (SIINFEKL disclosed as SEQ ID NO: 173)(open triangles) or with PBS (open squares) subcutaneously in the baseof the tail. A second and similar dose of immunogen was administeredeleven days after receiving the tumour cells. Animals were monitored fortumour incidence; animals were euthanased when tumour area exceeded 100mm².

FIG. 15 is a graphical representation showing therapeutic treatment ofLewis Lung tumour with a lipopeptide immunogen, as determined bymeasuring survival of animals following immunization. Mice were injectedwith 3×10⁴ Lewis Lung tumour cells that had been transfected withovalbumin and therefore expressed the CTL epitope SIINFEKL (SEQ ID NO:173) [Nelson et al., J. Immunol. 166: 5557-5566, 2001]. Four days afterreceiving tumour cells, animals were inoculated with 20 nmoles lipidatedpeptide [P25]-Lys(Pam₂Cys-Ser-Ser)SIINFEKL] (SIINFEKL disclosed as SEQID NO: 173) (open circles), non-lipidated peptide [P25]-Lys-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO: 173) (open triangles) or with PBS(open squares) subcutaneously in the base of the tail. A second andsimilar dose of immunogen was administered eleven days after receivingthe tumour cells. Animals were monitored for survival; animals wereeuthanased when tumour area exceeded 100 mm².

FIG. 16 is a graphical representation showing the ability of peptide andlipopeptide-based immunogens to up-regulate the expression of MHC classII, CD83 and CD86 on human dendritic cells. Human monocyte-deriveddendritic cells were incubated with media alone, LPS (5 pg/mL),non-lipidated peptide [P25]-Lys-[HCV] (5 μg/mL) or lipopeptide[P25]-Lys(Pam₂Cys-Ser-Ser)-[HCV] (5 μg/mL) for 48 hours before stainingwith FITC-conjugated antibodies for HLA-DR, CD83 and CD86 beforeanalysis by flow cytometry. Histograms are representative, of live largegranular cells gated on the forward and side scatter dot plot. Regionsof histograms shaded in grey and the given values correspond to thepercentage of cells that express high levels of antigen within theanalysed populations. The T helper cell epitope was identified fromMobillivirus and has the amino acid sequence KLIPNASLIENCTKAEL (SEQ IDNO: 20); the CTL epitope with the amino acid sequence DLMGYIPLV (SEQ IDNO: 176) is an HLA A2-restricted CTL epitope from the core protein ofhepatitis C virus.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Lipopeptides

One aspect of the invention provides an isolated lipopeptide comprisinga polypeptide conjugated to one or more lipid moieties wherein:

-   -   (i) said polypeptide comprises an amino acid sequence that        comprises:        -   (a) the amino acid sequence of a T helper cell (Th) epitope            and the amino acid sequence of a CTL epitope, wherein said            amino acid sequences are different; and        -   (b) one or more internal lysine residues or internal lysine            analog residues for covalent attachment of each of said            lipid moieties via the epsilon-amino group or terminal            side-chain group of said lysine or lysine analog; and    -   (ii) each of said one or more lipid moieties is covalently        attached directly or indirectly to an epsilon-amino group of        said one or more internal lysine residues or to a terminal        side-chain group of said internal lysine analog residues.

As used herein, the term “lipopeptide” means any non-naturally occurringcomposition of matter comprising one or more lipid moieties and one ormore amino acid sequences that are directly or indirectly conjugated,said composition of matter being substantially free of conspecificnon-conjugated lipid or protein.

By “directly” means that a lipid moiety and an amino acid sequence arejuxtaposed in said lipopeptide (i.e. they are not separated by a spacermolecule).

By “indirectly” means that a, lipid moiety and an amino acid sequenceare separated by a spacer comprising one or more carbon-containingmolecules, such as, for example, one or more amino acid residues. Theamino acid sequence may be of any length, constrained by the requirementfor functionality of both the T-helper epitope and the CTL epitope.

As used herein, the term “internal lysine residue” means a lysineresidue in the polypeptide comprising both the T-helper epitope and theCTL epitope, wherein said lysine is not the N-terminal amino acidresidue or the C-terminal residue of said polypeptide. Accordingly, theinternal lysine residue may be a C-terminal or N-terminal residue ofeither the T-helper epitope or the CTL epitope, provided that it isinternalized in the polypeptide. This means that the internal lysineresidue to which the lipid moiety is attached is a residue that ispresent in the amino acid sequence of the T helper cell epitope or theamino acid sequence of the CTL epitope. The internal lysine residue mayalso be distinct from the T-helper epitope or the CTL epitope, in whichcase it must link these two epitopes of the polypeptide.

Similarly, the term “internal lysine analog residue” means a lysineanalog residue in the polypeptide comprising both the T-helper epitopeand the CTL epitope, wherein said lysine analog is not the N-terminalamino acid residue or the C-terminal residue of said polypeptide. Thecriteria for establishing whether or not a lysine residue is “internal”shall apply mutatis mutandis to determining whether or not a lysineanalog is internal.

By “lysine analog” is meant a synthetic compound capable of beingincorporated into the internal part of a peptide that has a suitableside-group to which the lipid moiety can be coupled, including an aminoacid analog or non-naturally occurring amino acid having such an aminoside group. Preferred lysine analogs include compounds of the followinggeneral Formula (V):

wherein n is an integer from 0 to 3 and wherein X is a terminalside-chain group of said internal lysine analog residue selected fromthe group consisting of NH, O and S. More preferably, n is an integerhaving a value from 1 to 3. More preferably, X is an amino group. In aparticularly preferred embodiment, the lysine analog is selected fromthe group consisting of 2,3 diaminopropionic acid (Dpr),2,4-diaminobutyric acid (Dab) and 2,5-diaminovaleric acid [i.e.ornithine (Om)].

Those skilled in the art will know the meaning of the term“epsilon-amino group”. The term “terminal side-chain group” means asubstituent on the side chain of a lysine analog the is distal to thealpha-carbon of said analog, such as, for example, a beta-amino of Dpr,gamma-amino of Dab, or delta-amino of Om.

Preferably, the lipid moiety is attached via the epsilon amino group ofa lysine residue or to a terminal side-chain group of said internallysine analog residue that is positioned between the amino acidsequences of the T helper epitope and the CTL epitope.

The enhanced ability of the lipopeptides of the invention to elicit a Tcell response is reflected by their ability to upregulate the surfaceexpression of MHC class II molecules on immature dendritic cells (DC),particularly D1 cells, and by the enhanced number of CD8⁺ T cells intissue samples of immunized animals. In the case of animals immunizedusing CTLs of a viral pathogen, the enhanced ability of the lipopeptidesof the invention to elicit a T cell response is also indicated by theenhanced viral clearance following immunization of animals.

Preferably, the lipopeptides are soluble, more preferably highlysoluble.

As will be known to those skilled in the art, the epsilon amino group oflysine is the terminal amino group of the side chain of this amino acid.Use of the terminal side-chain group of the internal lysine or internallysine analog for cross-linkage to the lipid moiety facilitates thesynthesis of the polypeptide moiety as a co-linear amino acid sequenceincorporating both the T-helper epitope and the CTL epitope. There is aclear structural distinction between a lipopeptide having lipid attachedvia the epsilon amino group of a lysine residue or the terminalside-chain group of a lysine analog, and a lipopeptide having the lipidattached via an alpha amino group of a lysine in the peptide.

Accordingly, it is particularly preferred for at least one internallysine residue or internal lysine analog to which the lipid moiety isattached to be positioned within the polypeptide moiety so as toseparate the immunologically-functional epitopes. For example, theinternal lysine residue or internal lysine analog may act as a spacerand/or linking residue between the epitopes. Naturally, wherein theinternal lysine or internal lysine analog is positioned between theT-helper epitope and the CTL epitope, the lipid moiety will be attachedat a position that is also between these epitopes, albeit forming abranch from the amino acid sequence of the polypeptide. As exemplifiedherein, a single internal lysine residue is used to separate CTL andT-helper epitopes (eg. SEQ ID No: 4).

The present invention clearly contemplates the nesting of the internallysine residue or internal lysine analog residue within a third aminoacid sequence that does not function as a CTL epitope or T-helperepitope. For example, the internal lysine or internal lysine analog maybe conjugated to one or more different amino acid residues.

The epsilon amino group of the internal lysine or terminal side-chaingroup of an internal lysine analog can be protected by chemical groupswhich are orthogonal to those used to protect the alpha-amino andside-chain functional groups of other amino acids. In this way, theepsilon amino group or other side-chain group of an internal lysine orlysine analog can be selectively exposed to allow attachment of chemicalgroups, such as lipid-containing moieties, specifically to the epsilonamino group or side-chain amino group, as appropriate.

For peptide syntheses using Fmoc chemistry, a suitable orthogonallyprotected epsilon group of lysine is provided by the modified amino acidresidue Fmoc-Lys(Mtt)-OH (Nα-Fmoc-Nε-4-methyltrityl-L-lysine). Similarsuitable orthogonally-protected side-chain groups are available forvarious lysine analogs contemplated herein, eg. Fmoc-Om(Mtt)-OH(Nα-Fmoc-Nδ-4-methyltrityl-L-Ornithine), Fmoc-Dab(Mtt)-OH(Nα-Fmoc-Nγ4-methyltrityl-L-diaminobutyric acid) and Fmoc-Dpr(Mtt)-OH(Nα-Fmoc-Nβ-4-methyltrityl-L-diaminopropionic acid). The side-chainprotecting group Mtt is stable to conditions under which the Fmoc grouppresent on the alpha amino group of lysine or a lysine analog is removedbut can be selectively removed with 1% trifluoroacetate acid indichloromethane. Fmoc-Lys(Dde)-OH(Nα-Fmoc-Nε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine)or Fmoc-Lys(ivDde)-OH(Nα-Fmoc-Nε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine)can also be used in this context, wherein the Dde side-chain protectinggroups is selectively removed during peptide synthesis by treatment withhydrazine.

For peptide syntheses using Boc chemistry, Boc-Lys(Fmoc)-OH can be used.The side-chain protecting group Fmoc can be selectively removed bytreatment with piperidine or DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene)but remain in place when the Boc group is removed from the alphaterminus using trifluoroacetic acid.

Preferably, the T helper epitope and CTL epitope are separated by atleast one or two or three or four or five amino acid residues includinga single internal lysine residue or internal lysine analog residue.

The present invention clearly contemplates the addition of multiplelipid moieties to the polypeptide moiety. For example, the polypeptidemay include multiple internal lysine residues and/or multiple internallysine analogs. Steric hindrance may occur in the addition of lipid ifmultiple internal lysines or multiple lysine analogs are positioned moreclosely together, thereby producing a mixture of end-products, or areduced yield.

Relevant to this consideration is the fact that it is not necessary forthe entire amino acid sequence comprising the T-helper epitope or theentire amino acid sequence comprising the CTL epitope to have an immunefunction. Accordingly, the said amino acid sequences, whilst comprisingsaid epitopes may have additional sequence not possessing T-helper cellactivity or a CTL epitope. Where such additional sequences include oneor more internal lysine or lysine analog residues, the terminalside-chain groups of such residues may serve as attachment sites for thelipid moiety. Naturally, it is essential to retain both T-helperfunction and CTL epitope function.

The positioning of the internal lysine residue or internal lysine analogfor attachment of the lipid moiety should also be selected such thatattachment of the lipid moiety does not interfere with the immunefunction of the T-helper epitope or the CTL epitope in a subject to whomthe lipopeptide is administered. For example, depending upon theselection of lipid moiety, the attachment of said lipid within the CTLepitope may sterically hinder CTL epitope presentation.

A generalized preferred form of the lipopeptide of the invention,wherein the internal lysine or internal lysine analog is positionedbetween the T-helper and CTL epitopes is provided by the general Formula(VI).

wherein:

-   epitope is a T-helper epitope or CTL epitope;-   A is either present or absent and consists of an amino acid spacer    of about 1 to about 6 amino acids in length;-   n is an integer having a value of 1, 2, 3, or 4;-   X is a terminal side-chain group selected from the group consisting    of NH, O and S and preferably consisting of NH;-   Y is either present of absent and consists of an amino acid spacer    of about 1 to about 6 amino acids in length, wherein it is preferred    for said amino acid to be serine; and-   Z is a lipid moiety, preferably Pam₂Cys or Pam₃Cys.

The T-helper epitope is any T-helper epitope known to the skilledartisan for enhancing an immune response in a particular target subject(i.e. a human subject, or a specific non-human animal subject such as,for example, a rat, mouse, guinea pig, dog, horse, pig, or goat).Preferred T-helper epitopes comprise at least about 10-24 amino acids inlength, more generally about 15 to about 20 amino acids in length.

Promiscuous or permissive T-helper epitopes are particularly preferredas these are readily synthesized chemically and obviate the need to uselonger polypeptides comprising multiple T-helper epitopes.

Examples of promiscuous or permissive T-helper epitopes suitable for usein the lipopeptides of the present invention are selected from the groupconsisting of:

-   (i) a rodent or human T-helper epitope of tetanus toxoid peptide    (TTP), such as, for example amino acids 830-843 of TTP    (Panina-Bordignon et al., Eur. J. Immun. 19, 2237-2242, 1989);-   (ii) a rodent or human T-helper epitope of Plasmodium falciparum    pfg27;-   (iii) a rodent or human T-helper epitope of lactate dehydrogenase;-   (iv) a rodent or human T-helper epitope of the envelope protein of    HIV or HIVgp120 (Berzofsky et al., J. Clin. Invest. 88, 876-884,    1991);-   (v) a synthetic human T-helper epitope (PADRE) predicted from the    amino acid sequence of known anchor proteins (Alexander et al.,    Immunity 1, 751-761, 1994);-   (vi) a rodent or human T-helper epitope of measles virus fusion    protein (MV-F; Muller et al., Mol. Immunol. 32, 37-47, 1995;    Partidos et al., J. Gen. Virol., 71, 2099-2105, 1990);-   (vii) a T-helper epitope comprising at least about 10 amino acid    residues of canine distemper virus fusion protein (CDV-F) such as,    for example, from amino acid positions 148-283 of CDV-F (Ghosh et    al., Immunol. 104, 58-66, 2001; International Patent Publication No.    WO 00/46390);-   (viii) a human T-helper epitope derived from the peptide sequence of    extracellular tandem repeat domain of MUC1 mucin (US Patent    Application No. 0020018806);-   (ix) a rodent or human T-helper epitope of influenza virus    hemagglutinin (IV-H) (Jackson et al. Virol. 198, 613-623, 1994); and-   (x) a bovine or camel T-helper epitope of the VP3 protein of foot    and mouth disease virus (FMDV-01 Kaufbeuren strain), comprising    residues 173 to 176 of VP3 or the corresponding amino acids of    another strain of FMDV.

As will be known to those skilled in the art, a T-helper epitope may-berecognised by one or more mammals of different species. Accordingly, thedesignation of any T-helper epitope herein is not to be consideredrestrictive with respect to the immune system of the species in whichthe epitope is recognised. For example, a rodent T-helper epitope can berecognised by the immune system of a mouse, rat, rabbit, guinea pig, orother rodent, or a human or dog.

More preferably, the T-helper epitope will comprise an amino acidsequence selected from the group consisting of:

(SEQ ID NO: 1) (i) ALNNRFQIKGVELKS from IV-H; (SEQ ID NO: 14) (ii)GALNNRFQIKGVELKS from IV-H; (SEQ ID NO: 15) (iii)LSEIKGVIVHRLEGV from MV-F; (SEQ ID NO: 16) (iv)TAAQITAGIALHQSNLN from CDV-F; (SEQ ID NO: 17) (v)IGTDNVHYKIMTRPSHQ from CDV-F; (SEQ ID NO: 18) (vi)YKIMTRPSHQYLVIKLI from CDV-F; (SEQ ID NO: 19) (vii)SHQYLVIKLIPNASLIE from CDV-F; (SEQ ID NO: 20) (viii)KLIPNASLIENCTKAEL from CDV-F; (SEQ ID NO: 21) (ix)LIENCTKAELGEYEKLL from CDV-F; (SEQ ID NO: 22) (x)AELGEYEKLLNSVLEPI from CDV-F; (SEQ ID NO: 23) (xi)KLLNSVLEPINQALTLM from CDV-F; (SEQ ID NO: 24) (xii)EPINQALTLMTKNVKPL from CDV-F; (SEQ ID NO: 25) (xiii)TLMTKNVKPLQSLGSGR from CDV-F; (SEQ ID NO: 26) (xiv)KPLQSLGSGRRQRRFAG from CDV-F; (SEQ ID NO: 27) (xv)SGRRQRRFAGVVLAGVA from CDV-F; (SEQ ID NO: 28) (xvi)FAGWLAGVALGVATAA from CDV-F; (SEQ ID NO: 29) (xvii)GVALGVATAAQITAGIA from CDV-F; (SEQ ID NO: 30) (xviii)GIALHQSNLNAQAIQSL from CDV-F; (SEQ ID NO: 31) (xix)NLNAQAIQSLRTSLEQS from CDV-F; (SEQ ID NO: 32) (xx)QSLRTSLEQSNKAIEEI from CDV-F; (SEQ ID NO: 33) (xxi)EQSNKAIEEIREATQET from CDV-F; (SEQ ID NO: 34) (xxii)SSKTQTHTQQDRPPQPS from CDV-F; (SEQ ID NO: 35) (xxiii)QPSTELEETRTSRARHS from CDV-F; (SEQ ID NO: 36) (xxiv)RHSTTSAQRSTHYDPRT from CDV-F; (SEQ ID NO: 37) (xxv)PRTSDRPVSYTMNRTRS from CDV-F; (SEQ ID NO: 38) (xxvi)TRSRKQTSHRLKNIPVH from CDV-F; (SEQ ID NO: 39) (xxvii)TELLSIFGPSLRDPISA from CDV-F; (SEQ ID NO: 40) (xxviii)PRYIATNGYLISNFDES from CDV-F; (SEQ ID NO: 41) (xxix)CIRGDTSSCARTLVSGT from CDV-F; (SEQ ID NO: 42) (xxx)DESSCVFVSESAICSQN from CDV-F; (SEQ ID NO: 43) (xxxi)TSTIINQSPDKLLTFIA from CDV-F; (SEQ ID NO: 44) (xxxii)SPDKLLTFIASDTCPLV from CDV-F; (SEQ ID NO: 45) (xxxiii)STAPPAHGVTSAPDTRAPGSTAPP from MUC-1; (SEQ ID NO: 46) (xxxiv)GVTSAPDTRPAPGSTASSL from MUC-1; (SEQ ID NO: 47) (xxxv)GVTSAPDTRPAPGSTASL from MUC-1; (SEQ ID NO: 48) (xxxvi)TAPPAHGVTSAPDTRPAPGSTAPPKKG from MUC-1; (SEQ ID NO: 49) (xxxvii)STAPPAHGVTSAPDTRPAPGSTAPPK of MUC-1; (SEQ ID NO: 50) (xxxviii)GVAE from FMDV-VP3 protein; (SEQ ID NO: 51) (xxxix)TASGVAETTN of FMDV-VP3 (residues 170 to 179); and (SEQ ID NO: 52) (xl)TAKSKKFPSYTATYQF from FMDV.

The T-helper epitopes disclosed herein are included for the purposes ofexemplification only. Using standard peptide synthesis techniques knownto the skilled artisan, the T-helper epitopes referred to herein arereadily substituted for a different T-helper epitope to adapt thelipopeptide of the invention for use in a different species.Accordingly, additional T-helper epitopes known to the skilled person tobe useful in eliciting or enhancing an immune response in a targetspecies are not to be excluded.

Additional T-helper epitopes may be identified by a detailed analysis,using in vitro T-cell stimulation techniques of component proteins,protein fragments and peptides to identify appropriate sequences(Goodman and Sercarz, Ann. Rev. Immunol., 1, 465, (1983); Berzofsky, In“The Year in Immunology, Vol. 2” page 151, Karger, Basel, 1986; andLivingstone and Fathman, Ann. Rev. Immunol., 5, 477, (1987)).

The CTL epitope is conveniently derived from the amino acid sequence ofan immunogenic protein, lipoprotein, or glycoprotein of a virus,prokaryotic or eukaryotic organism, including but not limited to a CTLepitope derived from a mammalian subject or a bacterium, fungus,protozoan, or parasite that infects said subject. Mimotopes of the CTLepitopes are specifically included within the scope of the invention.

The CTL epitope will be capable of eliciting a T cell response whenadministered to a mammal, preferably by activating CD8+ T cells specificfor the epitope or antigen from which the epitope was derived, and morepreferably, by inducing cell mediated immunity against the pathogen ortumour cell from which the epitope is derived.

Shorter CTL epitopes are preferred, to facilitate peptide synthesis.Preferably, the length of the CTL epitope will not exceed about 30 aminoacids in length. More preferably, the CTL epitope sequence consists ofabout 25 amino acid residues or less, and more preferably less than 20amino acid residues, and even more preferably about 8-12 amino acidresidues in length.

Preferred CTL epitopes from parasites are those associated withleishmania, malaria, trypanosomiasis, babesiosis, or schistosomiasis,such as, for example a CTL epitope of an antigen of a parasite selectedfrom the group consisting of: Plasmodium falciparum; Circumsporozoa;Leishmania donovani; Toxoplasma gondii; Schistosoma mansoni; Schistosomajaponicum; Schistosoma hematobium; and Trypanosome brucei.

Particularly preferred CTL epitopes of P. falciparum are derived from anantigen selected from the group consisting of: circumsporozoite protein(CSP), sporozoite surface protein 2 (PfSSP2), liver stage antigen 1(LSA1), merozoite surface protein 1 (MSP1), serine repeat antigen(SERA), and AMA-1 antigen (Amante, et al. J. Immunol. 159, 5535-5544,1997; Chaba et al. Int. J. Immunopharm. 20, 259-273, 1998; Shi et al.,Proc. Natl. Acad. Sci. (USA) 96, 1615-1620, 1999; Wang et al. Science282, 476-479, 1998; and Zevering et al. Immunol. 94, 445-454, 1998).Particularly preferred CTL epitopes of L. donovani are derived from theRepetitive Peptide (Liew et al., J. Exp. Med. 172, 1359 (1990)).Particularly preferred CTL epitopes of T. gondii are derived from theP30 surface protein (Darcy et al., J. Immunol. 149, 3636 (1992)).Particularly preferred CTL epitopes of S. mansoni are derived from theSm-28GST antigen (Wolowxzuk et al., J. Immunol 146:1987 (1991)).

Preferred virus-specific CTL epitopes are derived from Rotaviruses,Herpes viruses, Corona viruses, Picornaviruses (eg. Apthovirus),Respiratory Synctial virus, Influenza Virus, Parainfluenza virus,Adenovirus, Pox viruses, Bovine herpes virus Type I, Bovine viraldiarrhea virus, Bovine rotaviruses, Canine Distemper Virus (CDV), Footand Mouth Disease Virus (FMDV), Measles Virus (MV), HumanImmunodeficiency Viruses (HIV), Feline Immunodeficiency Viruses (FIV),Epstein-Barr virus (EBV), Human Cytomegalovirus (HCMV), or hepatitisviruses, and the like.

Particularly preferred CTL epitopes of HIV-1 are derived from the env,gag, or pol proteins. Particularly preferred CTL epitopes of influenzavirus are derived from the nucleoprotein (Taylor et al., Immunogenetics26, 267 (1989); Townsend et al., Nature 348, 674 (1983)), matrix protein(Bednarek et al., J. Immunol. 147, 4047 (1991)) or polymerase protein(Jameson et al., J. Virol 72, 8682-8689, 1998; and Gianfrani et al.,Human Immunol. 61, 438-452, 2000). Particularly preferred CTL epitopesof Lymphocytic choriomeningitis virus (LCMV) are derived fromglycoprotein-1 antigen (Zinkarnagel et al. Nature 248, 701-702, 1974).Particularly preferred CTL epitopes of cytomegalovirus are derived froman antigen selected from the group consisting of: of pp28, pp50, pp65,pp71, pp150, gB, gH, 1E-1, 1E-2, US2, US3, US6, US11, and UL18 (eg.Diamond, U.S. Pat. No. 6,074,645, Jun. 13, 2000; Longmate et al.,Immunogenet. 52, 165-173, 2000; Wills et al., J. Virol. 70, 7569-7579,1996; Solache et al., J. Immunol. 163, 5512-5518, 1999; Diamond et al.,Blood 90, 1751-1767, 1997; Kern et al., Nature Med. 4, 975-978, 1998;Weekes et al., J. Virol. 73, 2099-2108, 1999; Retiêre et al., J. Virol.74, 3948-3952, 2000; and Salquin et al., Eur. J. Immunol. 30, 2531-2539,2000). Particularly preferred CTL epitopes of Measles Virus are derivedfrom the fusion glycoprotein (MV-F) and particularly from residues438-446 thereof (Herberts et al. J. Gen Virol. 82, 2131-2142, 2001).Particularly preferred epitopes from Epstein-Barr virus (EBV) arederived from a latent nuclear antigen (EBNA) or latent membrane protein(LMP) of EBV, such as, for example, EBNA 2A, EBNA 3A, EBNA 4A, or EBNA14a from EBV type A; EBNA 2B, EBNA 3B, EBNA 4B, or EBNA 14b from EBVtype B; LMP1; or LMP2 (International Patent Application No.PCT/AU95/00140 published Sep. 16, 1995; International Patent ApplicationNo. PCT/AU97/00328 published Nov. 24, 1997; and International PatentApplication No. PCT/AU98/00531 published Jan. 10, 1998).

Preferred bacteria-specific CTL epitopes are derived from Pasteurella,Actinobacillus, Haemophilus, Listeria monocytogenes, Mycobacteriumtuberculosis, Staphylococcus, Neisseria gonorrhoeae, Helicobacterpylori, Streptococcus pneumoniae, Salmonella enterica, E. coli,Shigella, and the like.

Suitable bacterial CTL epitopes include, for example, those CTL epitopesderived from the Mycobacterium tuberculosis 65 kd protein (Lamb et al.,EMBO J., 6, 1245 (1987)); M. tuberculosis ESAT-6 protein (Morten et al.,Infect. Immun. 66, 717-723, 1998); Staphylococcus aureus nucleaseprotein (Finnegan et al., J. Exp. Med. 164, 897 (1986)); Escherichiacoli heat stable enterotoxin (Cardenas et al., Infect. Immunity 61, 4629(1993)); and Escherichia coli heat labile enterotoxin (Clements et al.,Infect. Immunity 53, 685 (1986)).

Preferred CTL epitopes from mammalian subjects are derived from and/orcapable of generating T cell responses against a tumor CTL antigen.Tumor-specific CTL epitopes are usually native or foreign CTL epitopes,the expression of which is correlated with the development, growth,presence or recurrence of a tumor. In as much as such CTL epitopes areuseful in differentiating abnormal from normal tissue, they are usefulas a target for therapeutic intervention. Such CTL epitopes are wellknown in the art. Indeed, several examples are well-characterized andare currently the focus of great interest in the generation oftumor-specific therapies. Non-limiting examples of tumor CTL epitopesare derived from carcinoembryonic antigen (CEA), prostate specificantigen (PSA), melanoma antigen (MAGE, BAGE, GAGE), and mucins, such asMUC-1.

Preferred CTL epitopes for administering to a cancer patient are derivedfrom a protein that induces cancer, such as, for example, an oncoprotein(e.g., p53, ras etc.).

In a particularly preferred embodiment, the CTL epitope will comprise orconsist of an amino acid sequence selected from the group consisting of:

(SEQ ID NO: 2) (i) TYQRTRALV from the NP of PR8 virus; (SEQ ID NO: 53)(ii) KPKDELDYENDIEKKICKMEKCS of P.  falciparum CSP; (SEQ ID NO: 54)(iii) D1EKKICKMEKCSSVFNVVNS from P.  falciparum CSP; (SEQ ID NO: 55)(iv) KPIVQYDNF from P. falciparum LSA1; (SEQ ID NO: 56) (v)GISYYEKVLAKYKDDLE from P. falciparum  MSP1; (SEQ ID NO: 57) (vi)EFTYMINFGRGQNYWEHPYQKS of P. falciparum AMA-1; (SEQ ID NO: 58) (vii)DQPKQYEQHLTDYEKIKEG from P. falciparum AMA-1; (SEQ ID NO: 59) (viii)NMWQEVGKAM from HIV-1 env protein; (SEQ ID NO: 60) (ix)APTKAKRRVV from HIV-1 env protein; (SEQ ID NO: 61) (x)CTRPNNNTRK from HIV-1 env protein; (SEQ ID NO: 62) (xi)TVYYGVPVWK from HIV-1 env protein; (SEQ ID NO: 63) (xii)RPVVSTQLL from HIV-1 env protein; (SEQ ID NO: 64) (xiii)SLYNTVATLY from HIV-1 gag protein; (SEQ ID NO: 65) (xiv)ELRSLYNTVA from HIV-1 gag protein; (SEQ ID NO: 66) (xv)KIRLRPGGKK from HIV-1 gag protein; (SEQ ID NO: 67) (xvi)IRLRPGGKKK from HIV-1 gag protein; (SEQ ID NO: 68) (xvii)RLRPGGKKK from HIV-1 gag protein; (SEQ ID NO: 69) (xviii)GPGHKARVLA from HIV-1 gag protein; (SEQ ID NO: 70) (xix)SPIETVPVKL from HIV-1 pol protein; (SEQ ID NO: 71) (xx)ILKEPVHGVY from HIV-1 pol protein; (SEQ ID NO: 72) (xxi)AIFQSSMTK from HIV-1 pol protein; (SEQ ID NO: 73) (xxii)SPAIFQSSMT from HIV-1 pol protein; (SEQ ID NO: 74) (xxiii)QVRDQAEHLK from HIV-1 pol protein; (SEQ ID NO: 75) (xxiv)GPKVKQWPLT from HIV-1 pol protein; (SEQ ID NO: 76) (xxv)TYQRTRALV from influenza virus nucleoprotein; (SEQ ID NO: 77) (xvi)TYQRTRALVRTGMDP from influenza nucleoprotein; (SEQ ID NO: 78) (xvii)IASNENMDAMESSTL from influenza virus nucleoprotein; (SEQ ID NO: 79)(xxviii) KAVYNFATM from LCMV gp1; (SEQ ID NO: 80) (xxix)QVKWRMTTL from EBV; (SEQ ID NO: 81) (xxx) VFSDGRVAC from EBV;(SEQ ID NO: 82) (xxxi) VPAPAGPIV from EBV; (SEQ ID NO: 83) (xxxii)TYSAGIVQI from EBV; (SEQ ID NO: 84) (xxxiii) LLDFVRFMGV from EBV;(SEQ ID NO: 85) (xxxiv) QNGALAINTF from EBV; (SEQ ID NO: 86) (xxxv)VSSDGRVAC from EBV; (SEQ ID NO: 87) (xxxvi) VSSEGRVAC from EBV;(SEQ ID NO: 88) (xxxvii) VSSDGRVPC from EBV; (SEQ ID NO: 89) (xxxviii)VSSDGLVAC from EBV; (SEQ ID NO: 90) (xxxix) VSSDGQVAC from EBV;(SEQ ID NO: 91) (xl) VSSDGRVVC from EBV; (SEQ ID NO: 92) (xli)VPAPPVGPIV from EBV; (SEQ ID NO: 93) (xlii) VEITPYEPTG from EBV;(SEQ ID NO: 94) (xliii) VEITPYEPTW from EBV; (SEQ ID NO: 95) (xliv)VELTPYKPTW from EBV; (SEQ ID NO: 96) (xlv) RRIYDLIKL from EBV;(SEQ ID NO: 97) (xlvi) RKIYDLIEL from EBV; (SEQ ID NO: 98) (xlvii)PYLFWLAGI. from EBV; (SEQ ID NO: 99) (xlviii) TSLYNLRRGTALA from EBV;(SEQ ID NO: 100) (xlix) DTPLIPLTIF from EBV; (SEQ ID NO: 101) (l)TVFYN1PPMPL from EBV; (SEQ ID NO: 102) (li) VEITPYKPTW from EBV;(SEQ ID NO: 103) (lii) VSFIEFVGW from EBV; (SEQ ID NO: 104) (liii)FRKAQIQGL from EBV; (SEQ ID NO: 105) (liv) FLRGRAYGL from EBV;(SEQ ID NO: 106) (lv)  QAKWRLQTL from EBV; (SEQ ID NO: 107) (lvi)SVRDRLARL from EBV; (SEQ ID NO: 108) (lvii) YPLHEQHGM from EBV;(SEQ ID NO: 109) (lviii) HLAAQGMAY from EBV; (SEQ ID NO: 110) (lix)RPPIFIRRL from EBV; (SEQ ID NO: 111) (lx) RLRAEAGVK from EBV;(SEQ ID NO: 112) (lxi) IVTDFSVIK from EBV; (SEQ ID NO: 113) (lxii)AVFDRKSDAK from EBV; (SEQ ID NO: 114) (lxiii) NPTQAPVIQLVHAVY from EBV;(SEQ ID NO: 115) (lxiv) LPGPQVTAVLLHEES from EBV; (SEQ ID NO: 116) (lxv)DEPASTEPVHDQLL from EBV; (SEQ ID NO: 117) (lxvi) RYSIFFDY from EBV;(SEQ ID NO: 118) (lxvii) AVLLHEESM from EBV; (SEQ ID NO: 119) (lxviii)RRARSLSAERY from EBV; (SEQ ID NO: 120) (lxix) EENLLDFVRF from EBV;(SEQ ID NO: 121) (lxx) KEHVIQNAF from EBV; (SEQ ID NO: 122) (lxxi)RRIYDLIEL from.EBV; (SEQ ID NO: 123) (lxxii) QPRAPIRPI from EBV;(SEQ ID NO: 124) (lxxiii) EGGVGWRHW from EBV; (SEQ ID NO: 125) (lxxiv)CLGGLLTMV from EBV; (SEQ ID NO: 126) (lxxv) RRRWRRLTV from EBV;(SEQ ID NO: 127) (lxxvi) RAKFKQLL from EBV; (SEQ ID NO: 128) (lxxvii)RKCCRAKFKQLLQHYR from EBV; (SEQ ID NO: 129) (lxxviii)YLLEMLWRL from EBV; (SEQ ID NO: 130) (lxxvix) YFLEILWGL from EBV;(SEQ ID NO: 131) (lxxx) YLLEILWRL from EBV; (SEQ NO: 132) (lxxxi)YLQQNWWTL from EBV; (SEQ ID NO: 133) (lxxxii) LLLALLFWL from EBV;(SEQ ID NO: 134) (lxxxiii) LLVDLLWLL from EBV; (SEQ ID NO: 135) (lxxxiv)LLLIALWNL from EBV; (SEQ ID NO: 136) (lxxxv) WLLLFLAIL from EBV;(SEQ ID NO: 137) (lxxxvi) TLLVDLLWL from EBV; (SEQ ID NO: 138) (lxxxvii)LLWLLLFLA from EBV; (SEQ ID NO: 139) (lxxxviii) ILLIIALYL from EBV;(SEQ ID NO: 140) (lxxxix) VLFIFGCLL from EBV; (SEQ ID NO: 141) (xc)RLGATIWQL from EBV; (SEQ ID NO: 142) (xci) ILYFIAFAL from EBV;(SEQ ID NO: 143) (xcii) SLVIVTTFV from EBV; (SEQ ID NO: 144) (xciii)LMIIPLINV from EBV; (SEQ ID NO: 145) (xciv) TLFIGSHVV from EBV;(SEQ ID NO: 146) (xcv) LIPETVPYI from EBV; (SEQ ID NO: 147) (xcvi)VLQWASLAV from EBV; (SEQ ID NO: 148) (xcvii) QLTPHTKAV from EBV;(SEQ ID NO: 149) (xcviii) SVLGPISGHVLK from HCMV pp65; (SEQ ID NO: 150)(xcix) FTSQYRIQGKL from HCMV pp65; (SEQ ID NO: 151) (c)FVFPTKDVALR from HCMV pp65; (SEQ ID NO: 152) (ci)FPTKDVAL from HCMV pp65; (SEQ ID NO: 153) (cii)NLVPMVATV from HCMV pp65; (SEQ ID NO: 154) (ciii)MLNIPSINV from HCMV pp65; (SEQ ID NO: 155) (civ)RIFAELEGV from HCMV pp65; (SEQ ID NO: 156) (cv)TPRVTGGGGAM from HCMV pp65; (SEQ ID NO: 157) (cvi)RPHERNGFTVL from HCMV pp65; (SEQ ID NO: 158) (cvii)RLLQTGIHV from HCMV pp65; (SEQ ID NO: 159) (cviii)VIGDQYVKV from HCMV pp65; (SEQ ID NO: 160) (cix)ALFFFDIDL from HCMV pp65; (SEQ ID NO: 161) (cx)YSEHPTFTSQY from HCMV pp65; (SEQ ID NO: 162) (cxi)VLCPKNMII from HCMV pp65; (SEQ ID NO: 163) (cxii)DIYRIFAEL from HCMV pp65; (SEQ ID NO: 164) (cxiii)ILARNLVPMV from HCMV pp65; (SEQ ID NO: 165) (cxiv)EFFWDANDIY from HCMV pp65; (SEQ ID NO: 166) (cxv)IPSINVHHY) from HCMV pp65; (SEQ ID NO: 167) (cxvi)YILEETSVM from HCMV IE-1; (SEQ ID NO: 168) (cxvii)CVETMCNEY from HCMV IE-1; (SEQ ID NO: 169) (cxviii)RRIEEICMK from HCMV 1E-1; (SEQ ID NO: 170) (cxix)TTVYPPSSTAK from HCMV pp150; (SEQ ID NO: 171) (cxx)RRYPDAVYL from Measles Virus Fusion glycoprotein; (SEQ ID NO: 172)(cxxi) GYKDGNEYI from Listeria monocytogenes; (SEQ ID NO: 173) (cxxii)SIINFEKL from ovalbumin; and (SEQ ID NO: 176) (cxxiii)DLMGYIPLV from the core protein of hepatitis C virus.

It will be apparent from the preceding description that the polypeptidemoiety of the subject lipopeptide is synthesized conveniently as asingle amino acid chain, thereby requiring no post-synthesismodification to incorporate both epitopes. As exemplified herein, apolypeptide moiety comprising an amino acid sequence selected from thegroup consisting of the following is preferred:

(SEQ ID NO: 3) (i) ALNNRFQIKGVELKSTYQRTRALV; (SEQ ID NO: 4) (ii)ALNNRFQIKGVELKSKTYQRTRALV; (SEQ ID NO: 5) (iii)KLIPNASLIENCTKAELKTYQRTRALV; (SEQ ID NO: 6) (iv)KLIPNASLIENCTKAELKNLVPMVATV; (SEQ ID NO: 7) (v)AELGEYEKLLNSVLEPIKNLVPMVATV; (SEQ ID NO: 8) (vi)TAAQITAGIALHQSNLNKNLVPMVATV; (SEQ ID NO: 9) (vii)PRYIATNGYLISNFDESKNLVPMVATV; (SEQ ID NO: 10) (viii)KLIPNASLIENCTKAELKYLLEMLWRL; (SEQ ID NO: 11) (ix)AELGEYEKLLNSVLEPIKYLLEMLWRL; (SEQ ID NO: 12) (x)TAAQITAGIALHQSNLNKYLLEMLWRL; (SEQ ID NO: 13) (xi)PRYIATNGYLISNFDESKYLLEMLWRL; (SEQ ID NO: 174) (xii)KLIPNASLIENCTKAELKSIINFEKL; (SEQ ID NO: 175) (xiii)KLIPNASLIENCTKAELKGYKDGNEYI and (SEQ ID NO: 177) (xiv)KLIPNASLIENCTKAELKDLMGYIPLV.

For the purposes of nomenclature, SEQ ID Nos: 3-4 relate to syntheticpeptides comprising a T-helper epitope from the light chain of influenzavirus hemagglutinin (i.e. SEQ ID NO: 1) and an immunodominantH-2^(d)-restricted CTL epitope from the nucleoprotein of influenza virusstrain PR8 (i.e. SEQ ID NO: 2) wherein the internal lysine residue thatprovides a lipid attachment site at its epsilon-amino group is indicatedin bold type.

In SEQ ID No: 4, an additional internal lysine residue has beenengineered between the T-helper end CTL epitope (K16 in SEQ ID NO: 4).

SEQ ID No: 5 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 20) that isactive in dogs, mice, and humans and an immunodominantH-2^(d)-restricted CTL epitope from the nucleoprotein of influenza virusstrain PR8 (i.e. SEQ ID NO: 2) wherein the internal lysine residue thatprovides a lipid attachment site at its epsilon-amino group is indicatedin bold type. In this peptide, an additional internal lysine residue hasbeen engineered between the T-helper and CTL epitope (K18 in SEQ ID NO:5).

SEQ ID No: 6 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 20) that isactive in dogs, mice, and humans and an immunodominant HLA A2-restrictedCTL epitope from the immunodominant pp65 antigen of the cytomegalovirusof humans (i.e. HCMV pp65 antigen) (i.e. SEQ ID NO: 153) wherein theinternal lysine residue that provides a lipid attachment site at itsepsilon-amino group is indicated in bold type. In this peptide, anadditional internal lysine residue has been engineered between theT-helper and CTL epitope (K18 in SEQ ID NO: 6).

SEQ ID No: 7 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 22) that isactive in dogs, mice, and humans and an immunodominant HLA A2-restrictedCTL epitope from HCMV pp65 antigen (i.e. SEQ ID NO: 153) wherein theinternal lysine residue that provides a lipid attachment site at itsepsilon-amino group is indicated in bold type. In this peptide, anadditional internal lysine residue has been engineered between theT-helper and CTL epitope (K18 in SEQ ID NO: 7).

SEQ ID No: 8 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 16) that isactive in dogs, mice, and humans and an immunodominant HLA A2-restrictedCTL epitope from HCMV pp65 antigen (i.e. SEQ ID NO: 153) wherein theinternal lysine residue that provides a lipid attachment site at itsepsilon-amino group is indicated in bold type. In this peptide, anadditional internal lysine residue has been engineered between theT-helper and CTL epitope (K18 in SEQ ID NO: 8).

SEQ ID No: 9 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 40) that isactive in dogs, mice, and humans and an immunodominant HLA A2-restrictedCTL epitope from HCMV pp65 antigen (i.e. SEQ ID NO: 153) wherein theinternal lysine residue that provides a lipid attachment site at itsepsilon-amino group is indicated in bold type. In this peptide, anadditional internal lysine residue has been engineered between theT-helper and CTL epitope (K18 in SEQ ID. NO: 9).

SEQ ID No: 10 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 20) that isactive in dogs, mice, and humans and an immunodominant HLA A2-restrictedCTL epitope from Epstein-Barr virus LMP1 antigen (i.e. EBV LMP1; SEQ IDNO: 129) wherein the internal lysine residue that provides a lipidattachment site at its epsilon-amino group is indicated in bold type. Inthis peptide, an additional internal lysine residue has been engineeredbetween the T-helper and CTL epitope (K18 in SEQ ID NO: 10):

SEQ ID No: 11 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 22) that isactive in dogs, mice, and humans and an immunodominant HLA A2-restrictedCTL epitope from EBV LMP1 (SEQ ID NO: 129) wherein the internal lysineresidue that provides a lipid attachment site at its epsilon-amino groupis indicated in bold type. In this peptide, an additional internallysine residue has been engineered between the T-helper and CTL epitope(K18 in SEQ ID NO: 11).

SEQ ID No: 12 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 16) that isactive in dogs, mice, and humans and an immunodominant HLA A2-restrictedCTL epitope from EBV LMP1 (SEQ ID NO: 129) wherein the internal lysineresidue that provides a lipid attachment site at its epsilon-amino groupis indicated in bold type. In this peptide, an additional internallysine residue has been engineered between the T-helper and CTL epitope(K18 in SEQ ID NO: 12).

SEQ ID No: 13 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 40) that isactive in dogs, mice, and humans and an immunodominant HLA A2-restrictedCTL epitope from EBV LMP1 (SEQ ID NO: 129) wherein the internal lysineresidue that provides a lipid attachment site at its epsilon-amino groupis indicated in bold type. In this peptide, an additional internallysine residue has been engineered between the T-helper and CTL epitope(K18 in SEQ ID NO: 13).

SEQ ID No: 174 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 20) that isactive in dogs, mice, and humans and an immunodominant CTL epitope fromovalbumin (i.e. SEQ ID NO: 173) wherein the internal lysine residuesthat provide possible lipid attachment sites at its epsilon-amino groupare indicated in bold type. Preferably, the lipid is attached via K18 inSEQ ID NO: 174, which is an additional internal lysine residue that hasbeen engineered between the T-helper and CTL epitope.

SEQ ID No: 175 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 20) that isactive in dogs, mice, and humans and an immunodominant CTL epitope froma Listeria monocytogenes antigen (i.e. SEQ ID NO: 172) wherein theinternal lysine residues that provide possible lipid attachment sites atits epsilon-amino group are indicated in bold type. Preferably, thelipid is attached via K18 in SEQ ID NO: 175 which is an additionalinternal lysine residue that has been engineered between the T-helperand CTL epitope.

SEQ ID No: 177 relates to a synthetic peptide comprising a T-helperepitope from canine distemper virus (CDV-F; SEQ ID NO: 20) that isactive in dogs, mice, and humans and an immunodominant CTL epitope fromthe core protein of hepatitis C virus (SEQ ID NO: 176) wherein theinternal lysine residues that provide possible lipid attachment sites atits epsilon-amino group are indicated in bold type. Preferably, thelipid is attached via K18 in SEQ ID NO: 177.

The skilled artisan will readily be able to synthesize additionalpolypeptide moieties to those exemplified herein for use in the subjectlipopeptides, by substituting the T-helper epitope and/or the CTLepitope of anyone of SEQ ID Nos: 3-13, 174, 175 or 177 with anotherT-helper epitope or CTL epitope, such as, for example a T-helper epitopeset forth in any one of SEQ ID Nos: 14-52, or a CTL epitope set forth inany one of SEQ ID Nos: 53-173 or 176. Moreover, the selection ofappropriate T-helper epitope and CTL combinations will be apparent tothe skilled artisan from the disclosure provided herein, according tothe target species and the CTL epitope against which an immune responseis sought.

The amino acid sequences of the polypeptide moities described herein,including those exemplified polypeptides set forth in SEQ ID Nos: 3-13,174, 175 and 177 may be modified for particular purposes according tomethods well known to those of skill in the art without adverselyaffecting their immune function. For example, particular peptideresidues may be derivatized or chemically modified in order to enhancethe immune response or to permit coupling of the peptide to otheragents, particularly lipids. It also is possible to change particularamino acids within the peptides without disturbing the overall structureor CTL immunogenicity of the peptide. Such changes are therefore termed“conservative” changes and tend to rely on the hydrophilicity orpolarity of the residue. The size and/or charge of the side chains alsoare relevant factors in determining which substitutions areconservative.

It is well understood by the skilled artisan that, inherent in thedefinition of a biologically functional equivalent protein or peptide,is the concept that there is a limit to the number of changes that maybe made within a defined portion of the molecule and still result in amolecule with an acceptable level of equivalent biological activity.Biologically functional equivalent peptides are thus defined herein asthose peptides in which specific amino acids may be substituted.Particular embodiments encompass variants that have one, two, three,four, five or more variations in the amino acid sequence of the peptide.Of course, a plurality of distinct proteins/peptides with differentsubstitutions may easily be made and used in accordance with theinvention.

Those skilled in the art are well aware that the following substitutionsare permissible conservative substitutions (i) substitutions involvingarginine, lysine and histidine; (ii) substitutions involving alanine,glycine and serine; and (iii) substitutions involving phenylalanine,tryptophan and tyrosine. Peptides incorporating such conservativesubstitutions are defined herein as biologically functional equivalents.

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, J. MoL Biol. 157, 105-132, 1982). It is knownthat certain amino acids may be substituted for other amino acids havinga similar hydropathic index or score and still retain a similarbiological activity. The hydropathic index of amino acids also may beconsidered in determining a conservative substitution that produces afunctionally equivalent molecule. Each amino acid has been assigned ahydropathic index on the basis of their hydrophobicity and chargecharacteristics, as follows: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5). In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within. +/−0.2 is preferred. More preferably, thesubstitution will involve amino acids having hydropathic indices within+/−0.1, and more preferably within about +/−0.05.

It is also understood in the art that the substitution of like aminoacids is made effectively on the basis of hydrophilicity, particularlywhere the biological functional equivalent protein or peptide therebycreated is intended for use in immunological embodiments, as in thepresent case (e.g. U.S. Pat. No. 4,554,101). As detailed in U.S. Pat.No. 4,554,101, the following hydrophilicity values have been assigned toamino acid residues: arginine (+3.0); lysine (+3.0); aspartate(+3.0+/−0.1); glutamate (+3.0+/−0.1); serine (+0.3); asparagine (+0.2);glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5+/−0.1);alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3);valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); tryptophan (−3.4). In making changes based uponsimilar hydrophilicity values, it is preferred to substitute amino acidshaving hydrophilicity values within about +/−0.2 of each other, morepreferably within about +/−0.1, and even more preferably within about+/−0.05.

Having identified peptides suitable for use as immunogens, it also iscontemplated that other sterically similar compounds may be formulatedto mimic the key portions of the peptide structure. Such compounds,which may be termed peptidomimetics, may be used in the same manner asthe peptides of the invention and hence are also functional equivalents.The generation of a structural functional equivalent may be achieved bythe techniques of modeling and chemical design known to those of skillin the art. It will be understood that all such sterically similarconstructs fall within the scope of the present invention.

Another method for determining the “equivalence” of modified peptidesinvolves a functional approach. For example, a suitable variant peptidewill comprise an amino acid sequence that interacts at a significantlevel with a MHC Class I allele as determined using a predictivealgorithm for determining MHC Class I-binding epitopes, such as, forexample, the SYFPEITHI algorithm of the University of Tuebingen,Germany, or the algorithm of the HLA Peptide Binding Predictions programof the BioInformatics and Molecular Analysis Section (BIMAS) of theNational Institutes of Health of the Government of the United States ofAmerica. Such variant sequences will also bind to and/or stabilize anMHC Class I molecule on the surface of an APC (eg in the PBMC fractionor buffy coat fraction of serum) and/or will induce a memory CTLresponse or elicit IFN-γ production and/or will stimulate CTL activityin a standard cytotoxicity assay. The determination of suchfunctionalities is readily achievable by those skilled in the art.

The polypeptide moiety of the lipopeptide is readily synthesized usingstandard techniques, such as the Merrifield method of synthesis(Merrifield, J Am Chem Soc, 85:2149-2154, 1963) and the myriad ofavailable improvements on that technology (see e.g., Synthetic Peptides:A User's Guide, Grant, ed. (1992) W.H. Freeman & Co., New York, pp. 382;Jones (1994) The Chemical Synthesis of Peptides, Clarendon Press,Oxford, pp. 230.); Barany, G. and Merrifield, R. B. (1979) in ThePeptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284,Academic Press, New York; Wünsch, E., ed. (1974) Synthese von Peptidenin Houben-Weyls Methoden der Organischen Chemie (Müller, E., ed.), vol.15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984)Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky,M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis,Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. PeptideProtein Res. 25, 449-474.

The lipid moiety may comprise any C₂ to C₃₀ saturated, monounsaturated,or polyunsaturated linear or branched fatty acyl group, and preferably afatty acid group selected from the group consisting of: palmitoyl,myristoyl, stearoyl, lauroyl, octanoyl and decanoyl. Lipoamino acids areparticularly preferred lipid moieties within the present context. Asused herein, the term “lipoamino acid” refers to a molecule comprisingone or two or three or more lipids covalently attached to an amino acidresidue, such as, for example, cysteine or serine, lysine or an analogthereof. In a particularly preferred embodiment, the lipoamino acidcomprises cysteine and optionally, one or two or more serine residues.

The structure of the lipid moiety is not essential to activity of theresulting lipopeptide and, as exemplified herein, palmitic acid and/orcholesterol and/or Pam₁Cys and/or Pam₂Cys and/or Pam₃Cys can be used.The present invention clearly contemplates a range of other lipidmoieties for use in the lipopeptides, such as, for example, lauric acid,stearic acid or octanoic acid, without loss of immunogenicity.Accordingly, the present invention is not to be limited by the structureof the lipid moiety, unless specified otherwise, or the context requiresotherwise.

Similarly, the present invention is not to be limited by a requirementfor a single lipid moiety unless specified otherwise or the contextrequires otherwise. The addition of multiple lipid moieties to thepeptide moiety, such as, for example, to a position within the T-helperepitope, and to a position between the T-helper epitope and the B-cellepitope, is clearly contemplated.

The lipid moiety is preferably a compound having a structure of GeneralFormula (VII):

wherein:

-   (i) X is selected from the group consisting of sulfur, oxygen,    disulfide (—S—S—), and methylene (—CH₂—), and amino (—NH—);-   (ii) m is an integer being 1 or 2;-   (iii) n is an integer from 0 to 5;-   (iv) R₁ is selected from the group consisting of hydrogen, carbonyl    (—CO—), and R′—CO— wherein R′ is selected from the group consisting    of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25 carbon    atoms, and alkynyl having 7 to 25 carbon atoms, wherein said alkyl,    alkenyl or alkynyl group is optionally substituted by a hydroxyl,    amino, oxo, acyl, or cycloalkyl group;-   (v) R₂ is selected from the group consisting of R′—CO—O—, R′—O—,    R′—O—CO—; R′—NH—CO—, and R′—CO—NH—, wherein R′ is selected from the    group consisting of alkyl having 7 to 25 carbon atoms, alkenyl    having 7 to 0.25 carbon atoms, and alkynyl having 7 to 25 carbon    atoms, wherein said alkyl, alkenyl or alkynyl group is optionally    substituted by a hydroxyl, amino, oxo, acyl, or cycloalkyl group;    and-   (vi) R₃ is selected from the group consisting of R′—CO—O—, R′—O—,    R′-β-CO—, R′—NH—CO—, and R′—CO—NH—, wherein R′ is selected from the    group consisting of alkyl having 7 to 25 carbon atoms, alkenyl    having 7 to 25 carbon atoms, and alkynyl having 7 to 25 carbon    atoms, wherein said alkyl, alkenyl or alkynyl group is optionally    substituted by a hydroxyl, amino, oxo, acyl, or cycloalkyl group    and wherein each of R₁, R₂ and R₃ are the same or different.

Depending upon the substituent, the lipid moiety of general structure Vmay be a chiral molecule, wherein the carbon atoms directly orindirectly covalently bound to integers R₁ and R₂ are asymmetricdextrorotatory or levorotatory (i.e. an R or S) configuration.

Preferably, X is sulfur; m and n are both 1; R₁ is selected from thegroup consisting of hydrogen, and R′—CO—, wherein R′ is an alkyl grouphaving 7 to 25 carbon atoms; and R₂ and R₃ are selected from the groupconsisting of R′—CO—O—, R′—O—, R′—O—CO—, R′—NH—CO—, and R′—CO—NH—,wherein R′ is an alkyl group having 7 to 25 carbon atoms.

Preferably, R′ is selected from the group consisting of: palmitoyl,myristoyl, stearoyl and decanoyl. More preferably, R′ is palmitoyl.

Each integer R′ in said lipid moiety may be the same or different.

In a particularly preferred embodiment, X is sulfur; m and n are both 1;R₁ is hydrogen or R′—CO— wherein R′ is palmitoyl; and R₂ and R₃ are eachR′—CO—O— wherein R′ is palmitoyl. These particularly preferred compoundsare shown by Formula (I) and Formula (II) supra.

The lipid moiety can also have the following General Formula (VIII):

wherein:

-   (i) R₄ is selected from the group consisting of: (i) an    alpha-acyl-fatty acid residue consisting of between about 7 and    about 25 carbon atoms; (ii) an alpha-alkyl-beta-hydroxy-fatty acid    residue; (iii) a beta-hydroxy ester of an    alpha-alkyl-beta-hydroxy-fatty acid residue wherein the ester group    is preferably a straight chain or branched chain comprising more    than 8 carbon atoms; and (iv) a lipoamino acid residue; and-   (ii) R₅ is hydrogen or the side chain of an amino acid residue.

Preferably, R₄ consists of between about 10 and about 20 carbon atoms,and more preferably between about 14 and about 18 carbon atoms.

Optionally, wherein R₄ is a lipoamino acid residue, the side-chain ofthe integers R₄ and R₅ can form a covalent linkage. For example, whereinR₄ comprises an amino acid selected from the group consisting of lysine,ornithine, glutamic acid, aspartic acid, a derivative of lysine, aderivative of ornithine, a derivative of glutamic acid, and a derivativeof aspartic acid, then the side chain of that amino acid or derivativeis covalently attached; by virtue of an amide or ester linkage, to R₅.

Preferably, the structure set forth in General Formula VIII is a lipidmoiety selected from the group consisting of: N,N′-diacyllysine;N,N′-diacylornithine; di(monoalkyl)amide or ester of glutamic acid;di(monoalkyl)amide or ester of aspartic acid; a N,O-diacyl derivative ofserine, homoserine, or threonine; and a N,S-diacyl derivative ofcysteine or homocysteine.

Amphipathic molecules, particularly those having a hydrophobicity notexceeding the hydrophobicity of Pam₃Cys (Formula (I)) are alsopreferred.

The lipid moieties of Formula (I), Formula (II), Formula (VI) or Formula(VIII) are further modified during synthesis or post-synthetically, bythe addition of one or more spacer molecules, preferably a spacer thatcomprises carbon, and more preferably one or more amino acid residues.These are conveniently added to the lipid structure via the terminalcarboxy group in a conventional condensation, addition, substitution, oroxidation reaction. The effect of such spacer molecules is to separatethe lipid moiety from the polypeptide moiety and increase immunogenicityof the lipopeptide product.

Serine dimers, trimers, tetramers, etc, are particularly preferred forthis purpose.

Preferably, such spacers include a terminal protected amino acid residueto facilitate the later conjugation of the modified lipoamino acid tothe polypeptide.

Exemplary modified lipoamino acids produced according to this embodimentare presented as Formulae (III) and (IV), which, are readily derivedfrom Formulae (I) and (II), respectively by the addition of a serinehomodimer. As exemplified herein, Pam₃Cys of Formula (I), or Pam₂Cys ofFormula (II) is conveniently synthesized as the lipoamino acidsPam₃Cys-Ser-Ser of Formula (III), or Pam₂Cys-Ser-Ser of Formula (IV) forthis purpose.

As an alternative to the addition of a spacer to the lipid moiety, thespacer may be added to the epsilon amino group of the internal lysineresidue or to the terminal side-chain group of a lysine analog in thepolypeptide moiety, either as a short peptide, such as, for example aserine homodimer, homotrimer, homotetramer, etc, or alternatively, bythe sequential addition of amino acid residues, thereby producing abranched polypeptide chain. This approach takes advantage of themodified nature of the terminal side-chain group on the internal lysineor lysine analog to achieve specificity in the addition of the spacer.Naturally, to avoid sequential spacer addition, the terminal amino acidresidue of the spacer should preferably be protected, such thatde-protection can facilitate conjugation of the lipid moiety to thebranched polypeptide.

Alternatively, the spacer may be added to a non-modified epsilon aminogroup of the polypeptide by conventional nucleophilic substitutionreaction. However, it is preferred to follow this approach if thepolypéptide has an amino acid sequence comprising a single internallysine residue and a blocked N-terminus.

The lipid moiety is prepared by conventional synthetic means, such as,for example, the methods described in U.S. Pat. Nos. 5,700,910 and6,024,964, or alternatively, the method described by Wiesmuller et al,Hoppe Seylers Zur Physiol. Chem. 364, 593 (1983), Zeng et al., J. Pept.Sci 2, 66 (1996); Jones et al., Xenobiotica 5, 155 (1975), or Metzger etal., Int. J. Pept. Protein Res. 38, 545 (1991). Those skilled in the artwill be readily able to modify such methods to achieve the synthesis ofa desired lipid for use conjugation to a polypeptide.

Combinations of different lipids are also contemplated for use in thelipopeptides of the invention. For example, one or twomyristoyl-containing lipids or lipoamino acids are attached via internallysine or lysine analog residues to the polypeptide moiety, optionallyseparated from the polypeptide by a spacer, with one or twopalmitoyl-containing lipid or lipoamino acid molecules attached tocarboxy terminal lysine amino acid residues. Other combinations are notexcluded.

The lipopeptides of the invention are readily modified for diagnosticpurposes. For example, it is modified by addition of a natural orsynthetic hapten, an antibiotic, hormone, steroid, nucleoside,nucleotide, nucleic acid, an enzyme, enzyme substrate, an enzymeinhibitor, biotin, avidin, polyethylene glycol, a peptidic polypeptidemoiety (e.g. tuftsin, polylysine), a fluorescence marker (e.g. FITC,RITC, dansyl, luminol or coumarin), a bioluminescence marker, a spinlabel, an alkaloid, biogenic amine, vitamin, toxin (e.g. digoxin,phalloidin, amanitin, tetrodotoxin), or a complex-forming agent.

As exemplified herein, highly immunogenic lipopeptides capable ofinducing CTL responses are provided, said lipopeptides comprisingPam₃Cys of Formula (I), or Pam₂Cys of Formula (II) conjugated via theepsilon amino group of a lysine residue positioned between the CD4+T-helper epitope and a CD8⁺ CTL epitope.

Preparation of Lipopeptides

A second aspect of the invention provides a method of producing alipopeptide comprising:

-   (i) producing a polypeptide comprising an amino acid sequence that    comprises:    -   (a) the amino acid sequence of a T helper cell (Th) epitope and        the amino acid sequence of a CTL epitope, wherein said amino        acid sequences are different; and    -   (b) one or more internal lysine residues or internal lysine        analog residues; and-   (iii) covalently attaching each of said one or more lipid moieties    directly or indirectly to an epsilon-amino group of said one or more    internal lysine residues or to the terminal side-chain group of said    one or more internal lysine analog residues so as to produce a    lipopeptide having the lipid moiety attached to the epsilon amino    group of said internal lysine residue or having the lipid moiety    attached to the terminal side-chain group of said internal lysine    analog residue.

Preferably, the method further comprises production of the lipid moiety.

Conventional chemical syntheses referred to herein are the preferredmeans for producing the polypeptide moiety and the lipid moiety.

Preferably, the internal lysine or lysine analog is modified byselective removal of a blocking group (eg: Mtt) from the terminalside-chain group so as to permit the addition of an amino acid residue,a spacer or lipid moiety, including a lipoamino acid, at that position.

For attachment of the lipid to the polypeptide, it is convenient for thefunctional groups of the polypeptide to be protected in a manner knownin the art of peptide synthesis, to ensure that no undesirable reactionsat those groups takes place at a significant reaction rate.

By known coupling processes, the polypeptide is synthesized on a solidor soluble carrier, such as a polymer (for example Merrifield resin) andmade available for conjugation to a spacer, amino acid, or lipid. Forexample, the terminal side chain group of the lysine or lysine analog(eg. epsilon amino group of the internal lysine) is protected by one ofa number of protecting groups. Blocking groups (also called protectinggroups or masking groups) are used to protect the amino group of theamino acid having an activated carboxyl group that is involved in thecoupling reaction, or to protect the carboxyl group of the amino acidhaving an acylated amino group that is involved in the couplingreaction. For coupling to occur, a blocking group must be removedwithout disrupting a peptide bond, or any protecting group attached toanother part of the peptide.

For solid phase peptide synthesis, blocking groups that are stable tothe repeated treatments necessary for removal of the amino blockinggroup of the growing peptide chain and for repeated amino acidcouplings, are used for protecting the amino acid side-chains.Additionally, the peptide-resin anchorage that protects the C-terminusof the peptide must be protected throughout the synthetic process untilcleavage from the resin is required. Accordingly, by the judiciousselection of orthogonally protected alpha-amino acids, lipids and/oramino acids are added at desired locations to a growing peptide whilstit is still attached to the resin.

Preferred amino blocking groups are easily removable but sufficientlystable to survive conditions for the coupling reaction and othermanipulations, such as, for example, modifications to the side-chaingroups. Preferred amino blocking groups are selected from the groupconsisting of: (i) a benzyloxycarbonyl group (Z or carbobenzoxy) that isremoved easily by catalytic hydrogenation at room temperature andordinary pressure, or using sodium in liquid ammonia and hydrobromicacid in acetic acid; (ii) a t-Butoxycarbonyl group (Boc) that isintroduced using t-butoxycarbonyl azide or di-tert-butyldicarbonate andremoved using mild acid such as, for example, trifluoroacetic acid (50%TFA in dichloromethane), or HCI in acetic acid/dioxane/ethylacetate;(iii) a 9-fluorenylmethyloxycarbonyl group (Fmoc) that is cleaved undermildly basic, non-hydrolytic conditions, such as, for example, using aprimary or secondary amine (eg. 20% piperidine in dimethyl formamide);(iv) a 2-(4-biphenylyl) propyl(2)oxycarbonyl group (Bpoc); (v) a2-nitro-phenylsulfenyl group (Nps); and (vi) a dithia-succinoyl group(Dts).

Side chain-protecting groups will vary for the functional side chains ofthe amino acids forming the peptide being synthesized. Side-chainprotecting groups are generally based on the Bzl group or the tBu group.Amino acids having alcohols or carboxylic acids in the side-chain areprotected as Bzl ethers, Bzl esters, cHex esters, tBu ethers, or tBuesters. Side-chain protection of Fmoc amino acids requires blockinggroups that are ideally base stable and weak acid (TFA) labile. Forexample, the epsilon-amino group of Lysine is protected using Mtt (eg.Fmoc-lysine(Mtt)-OH). Alternatively, a halogenated benzyl derivativesuch as CIZ is used to protect the lysine side chain should enhancedacid stability be required. The thiol group of Cystine, the imidazole ofHistidine, or guanidino group of Arginine, generally require specialisedprotection. Many different protecting groups for peptide synthesis havebeen described (see The Peptides, Gross et al. eds., Vol. 3, AcademicPress, New York, 1981).

The two most widely used protection strategies are the Boc/Bzl- and theFmoc/tBu-strategies. In Boc/Bzl, Boc is used for amino protection andthe side-chains of the various amino acids are protected using Bzl- orcHex-based protecting groups. A Boc group is stable under catalytichydrogenation conditions and is used orthogonally along with a Z groupfor protection of many side chain groups. In Fmoc/tBu, Fmoc is used foramino protection and the side-chains are protected with tBu-basedprotecting groups.

Peptides are lipidated by methods well known in the art. Standardcondensation, addition, substitution or oxidation (e.g. disulfide bridgeformation or amide bond formation between a terminal amino group on theinternal lysine or lysine analog with the carboxy terminal group of anincoming amino acid or peptide or lipoamino acid) reactions result inthe addition of lipid to the polypeptide.

In an alternative embodiment, a peptide of the present invention for useas an immunogen is produced by chemoselective ligation or chemicalconjugation or oxime chemistry. Such methods are well-known in the art,and allow for the individual peptide components to be produced bychemical or recombinant means, followed by their chemoselective ligationin an appropriate configuration or conformation or order (eg. Nardin etal., Vaccine 16, 590 (1998); Nardin et al., J. Immunol. 166, 481 (2001);Rose et al., Mol. Immunol. 32, 1031 (1995); Rose et al., Bioconjug. Chem7, 552 (1996); and Zeng et al., Vaccine 18, 1031 (2000), which areincorporated herein by reference).

Lipopeptide Formulations

The lipopeptide is conveniently formulated in a pharmaceuticallyacceptable excipient or diluent, such as, for example, an aqueoussolvent, non-aqueous solvent, non-toxic excipient, such as a salt,preservative, buffer and the like. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oil and injectableorganic esters such as ethyloleate. Aqueous solvents include water,alcoholic/aqueous solutions, saline solutions, parenteral vehicles suchas sodium chloride, Ringer's dextrose, etc. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. The pHand exact concentration of the various components the pharmaceuticalcomposition are adjusted according to routine skills in the art.

The addition of an extrinsic adjuvant to the lipopeptide formulation,although generally not required, is also encompassed by the invention.Such extrinsic adjuvants include all acceptable immunostimulatorycompounds such as, for example, a cytokine, toxin, or syntheticcomposition. Exemplary adjuvants include IL-1, IL-2, BCG, aluminumhydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP) 1983A, referred to as MTP-PE), lipid A, MKL and RIBI, whichcontains three components extracted from bacteria, monophosphoryl lipidA, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%squalene/Tween 80 emulsion.

It may be desirable to co-administer biologic response modifiers (BRM)with the lipopeptide, to down regulate suppressor T cell activity.Exemplary BRM's include, but are not limited to, Cimetidine (CIM; 1200mg/d) (Smith/Kline, PA, USA); Indomethacin (IND; 150 mg/d) (Lederle,N.J., USA); or low-dose Cyclophosphamide (CYP; 75, 150 or 300mg/m.sup.2) (Johnson/Mead, N.J., USA).

Use of the Lipopeptide in Immunization

The novel lipopeptides of the invention differ in essential aspects fromknown lipopeptide conjugates of CTL epitopes in having the lipid moietyconjugated exclusively through the terminal side-chain group of aninternal lysine or lysine analog residue, thereby enhancing T cellresponses without the administration of additional adjuvant.Accordingly, a particular utility of the lipopeptides of the presentinvention is in the fields of eliciting a T cell response either in vivoor ex vivo, synthetic vaccine preparation, diagnostic methods employingT cells, and immunotherapy for veterinary and human medicine.

More particularly, the lipopeptide of the present invention enhances CTLmemory responses against the CTL epitope moiety when administered to ananimal subject, without any requirement for an adjuvant to achieve asimilar level of CTL activation. In addition, enhanced maturation ofdendritic cells and other biological effects which include induction ofIFN-γ producing CD8+ cells as well as viral, bacterial and tumour cellclearance have been observed following administration of vaccine:

Accordingly, a further aspect of the invention provides a method ofenhancing cell mediated immunity against the organism from which the CTLepitope is derived in a subject comprising administering the lipopeptideof the invention or a derivative or a functionally equivalent variant ofsaid lipopeptide or a vaccine composition comprising said lipopeptide orvariant or derivative for a time and under conditions sufficient toactivate a CTL and/or a CTL precursor of said subject.

By “CTL precursor” is meant a naive T cell (ie. a T cell that expressesone or more T cell receptors on its surface and is capable ofproliferating and differentiating into a memory T cell or effector Tcell).

Preferably, the lipopeptide or vaccine is administered prophylacticallyto a subject not harboring a latent or active infection by a parasite,bacterium or virus, or suffering from a cancer or administeredtherapeutically to a subject harboring a latent or active infection by aparasite, bacteria or Virus or suffering from a cancer. In the presentcontext, the term “activate” means that the ability of a T cell torecognize and lyse a cell harboring an antigen from which the CTLepitope is derived is enhanced, or that the ability of a T cell torecognize a T cell epitope of said antigen is enhanced, eithertransiently or in a sustained manner. The term “activate” shall also betaken to include a reactivation of a T cell population followingactivation of a latent infection by a parasite or bacteria or virus, orfollowing re-infection with a parasite or bacteria or virus, orfollowing immunization of a previously-infected subject with alipopeptide or composition of the invention.

Those skilled in the art are aware that optimum T cell activationrequires cognate recognition of antigen/MHC by the T cell receptor(TcR), and a co-stimulation involving the ligation of a variety of cellsurface molecules on the T cell with those on an antigen presenting cell(APC). The costimulatory interactions CD28/B7, CD40L/CD40 and OX40/0X40Lare preferred, but not essential for T cell activation. Othercostimulation pathways may operate.

For determining the activation of a CTL or precursor CTL or the level ofepitope-specific activity, standard methods for assaying the number ofCD8⁺ T cells in a specimen can be used. Preferred assay formats includea cytotoxicity assay, such as for example the standard chromium releaseassay, the assay for IFN-γ production; such as, for example, the ELISPOTassay. These assay formats are described in detail in the accompanyingexamples.

MHC class ⁻1 Tetramer assays can also be utilized, particularly for CTL.Epitope-specific quantitation of CD8⁺ T cells (Altman et al., Science274, 94-96, 1996; Ogg et al., Curr Opin Immunol. 10, 393-396, 1998). Toproduce tetramers, the carboxyl terminus of an MHC molecule, such as,for example, the HLA A2 heavy chain, is associated with a specificpeptide epitope or polyepitope, and treated so as to form a tetramercomplex having bound hereto a suitable reporter molecule, preferably afluorochrome such as, for example, fluoroscein isothiocyanate (FITC),phycoerythrin, phycocyanin or allophycocyanin. Tetramer formation isachieved, for example, by producing the MHC-peptide fusion protein as abiotinylated molecule and then mixing the biotinylated MHC-peptide withdeglycosylated avidin that has been labeled with a fluorophore, at amolar ratio of 4:1. The Tetramers produced bind to a distinct set ofCD8⁺ T cell receptors (TcRs) on a subset of CD8⁺ T cells derived fromthe subject (eg in whole blood or a PBMC sample), to which the peptideis HLA restricted. There is no requirement for in vitro T cellactivation or expansion. Following binding, and washing of the T cellsto remove unbound or non-specifically bound Tetramer, the number of CD8⁺cells binding specifically to the HLA-peptide Tetramer is readilyquantified by standard flow cytometry methods, such as, for example,using a FACSCalibur Flow cytometer (Becton Dickinson). The Tetramers canalso be attached to paramagnetic particles or magnetic beads tofacilitate removal of non-specifically bound reporter and cell sorting.Such particles are readily available from commercial sources (eg.Beckman Coulter, Inc., San Diego, Calif., USA) Tetramer staining doesnot kill the labeled cells; therefore cell integrity is maintained forfurther analysis. MHC Tetramers enable the accurate quantitativeanalyses of specific cellular immune responses, even for extremely rareevents that occur at less than 1% of CD8⁺ T cells (Bodinier et al.,Nature Med. 6, 707-710, 2000; Ogg et al., Curr Opin Immunol. 10,393-396, 1998).

The total number of CD8⁺ cells in a sample can also be determinedreadily, such as, for example, by incubating the sample with amonoclonal antibody against CD8 conjugated to a different reportermolecule to that used for detecting the Tetramer. Such antibodies arereadily available (eg. Becton Dickinson). The relative intensities ofthe signals from the two reporter molecules used allows quantificationof both the total number of CD8⁺ cells and Tetramer-bound T cells and adetermination of the proportion of total T cells bound to the Tetramer.

Because CD4⁺ T-helper cells function in CMI as producers of cytokines,such as, for example IL-2, to facilitate the expansion of CD8⁺ T cellsor to interact with the APC thereby rendering it more competent toactivate CD8⁺ T cells, cytokine production is an indirect measure of Tcell activation. Accordingly, cytokine assays can also be used todetermine the activation of a CTL or precursor CTL or the level of cellmediated immunity in a human subject. In such assays, a cytokine suchas, for example, IL-2, is detected or production of a cytokine isdetermined as an indicator of the level of epitope-specific reactive Tcells.

Preferably, the cytokine assay format used for determining the level ofa cytokine or cytokine production is essentially as described byPetrovsky and Harrison, J. Immunol. Methods 186, 37-46, 1995, whichassay reference is incorporated herein.

Preferably, the cytokine assay is performed on whole blood or PBMC orbuffy coat.

Preferably, the lipopeptide or derivative or variant or vaccinecomposition is administered for a time and under conditions sufficientto elicit or enhance the expansion of CD8⁺ T cells.

Still more preferably, the lipopeptide or derivative or variant orvaccine composition is administered for a time and under conditionssufficient for cell mediated immunity (CMI) to be enhanced in thesubject.

By “CMI” is meant that the activated and clonally expanded CTLs areMHC-restricted and specific for a CTL epitope. CTLs are classified basedon antigen specificity and MHC restriction, (ie., non-specific CTLs andantigen-specific, MHC-restricted CTLs). Non-specific CTLs are composedof various cell types, including NK cells and can function very early inthe immune response to decrease pathogen load, while antigen-specificresponses are still being established. In contrast, MHC-restricted CTLsachieve optimal activity later than non-specific CTL, generally beforeantibody production. Antigen-specific CTLs inhibit or reduce the spreadof a pathogen and preferably terminate infection.

CTL activation, clonal expansion, or CMI can be induced systemically orcompartmentally localized. In the case of compartmentally localizedeffects, it is preferred to utilize a vaccine composition suitablyformulated for administration to that compartment. On the other hand,there are no such stringent requirements for inducing CTL activation,expansion or CMI systemically in the subject.

The effective amount of lipopeptide to be administered, either solus orin a vaccine composition to elicit CTL activation, clonal expansion orCMI will vary, depending upon the nature of the immunogenic epitope, theroute of administration, the weight, age, sex, or general health of thesubject immunized, and the nature of the CTL response sought. All suchvariables are empirically determined by art-recognized means.

The lipopeptide, optionally formulated with any suitable or desiredcarrier, adjuvant, BRM, or pharmaceutically acceptable excipient, isconveniently administered in the form of an injectable composition.Injection may be intranasal, intramuscular, sub-cutaneous, intravenous,intradermal, intraperitoneal, or by other known route. For intravenousinjection, it is desirable to include one or more fluid and nutrientreplenishers.

The optimum dose to be administered and the preferred route foradministration are established using animal models, such as, forexample, by injecting a mouse, rat, rabbit, guinea pig, dog, horse, cow,goat or pig, with a formulation comprising the lipopeptide, and thenmonitoring the CTL immune response using any conventional assay.

The use of HLA A2/K^(b) transgenic mice carrying a chimeric human-mouseClass I major histocompatibility complex (MHC) locus composed of the α1and α2 domains of the human HLA A*0201 allele and the α3 domain of themouse H-2K^(b) Class I molecules (Vitiello et al., J. Exp. Med. 173,1007, 1991) is particularly preferred for testing responses in vivo to alipopeptide of the invention that comprises a HLA A2-restricted CTLepitope or a vaccine composition comprising same.

Without being bound by any theory or mode of action, we believe that thebiological effects of the lipopeptides are exerted through their abilityto stimulate and mature dendritic cells. It is the dendritic cells whichthen activate CD4+ and CD8+ T cells in the draining lymph nodes. Forthis reason, we would not nor would it be possible to activate T cells:directly as envisaged. The following section has therefore been modifiedaccordingly to accommodate the notion of dendritic cell activation.

In a related embodiment, the invention provides a method of enhancingthe cell mediated immunity of a subject, said method comprisingcontacting ex vivo cells, preferably dendritic cells, obtained from asubject with an immunologically active lipopeptide of the invention or aderivative or variant thereof or a vaccine composition comprising saidlipopeptide or derivative or variant for a time and under conditionssufficient to mature said dendritic cells. Said dendritic cells are thencapable of conferring epitope specific activation of T cells.

In a preferred embodiment, the invention provides a method of enhancingthe cell mediated immunity of a subject, said method comprising:

-   (i) contacting ex vivo dendritic cells obtained from a subject with    an immunologically active lipopeptide of the invention or a    derivative or variant thereof or a vaccine composition comprising    said lipopeptide or derivative or variant for a time and under    conditions sufficient to mature said dendritic cells; and-   (ii) introducing the activated dendritic cells autologously to the    subject or syngeneically to another subject in order that T cell    activation occurs.

The T cell may be a CTL or CTL precursor cell.

The subject from whom the dendritic cells are obtained may be the samesubject or a different subject to the subject being treated. The subjectbeing treated can be any subject carrying a latent, or active infectionby a pathogen, such as, for example, a parasite, bacterium or virus or asubject who is otherwise in need of obtaining vaccination against such apathogen or desirous of obtaining such vaccination. The subject beingtreated may also be treated for a tumour that they are carrying.

By “epitope specific activity” is meant that the T cell is renderedcapable of being activated as defined herein above (ie. the T cell willrecognize and lyze a cell harboring a pathogen from which the CTLepitope is derived, or is able to recognize a T cell epitope of anantigen of a pathogen either transiently or in a sustained manner).Accordingly, it is particularly preferred for the T cell to be a CTLprecursor which by the process of the invention is rendered able torecognize and lyze a cell harboring the pathogen or able to recognize aT cell epitope of an antigen of the pathogen either transiently or in asustained manner.

For such an ex vivo application; the dendritic cells are preferablycontained in a biological sample obtained from a subject, such as, forexample, blood, PBMC or a buffy coat fraction derived therefrom.

Another aspect of the invention provides a method of providing orenhancing immunity against a pathogen in an uninfected subjectcomprising administering to said subject an immunologically activelipopeptide of the invention or a derivative or variant thereof or avaccine composition comprising said lipopeptide or derivative or variantfor a time and under conditions sufficient to provide immunologicalmemory against a future infection by the pathogen. As with the otherembodiments described herein, the pathogen may be a parasite, virus orbacterium, and is preferably a parasite, virus or bacterium referred toherein above from which a CTL epitope has been identified.

In a related embodiment, the invention provides a method of enhancing orconferring immunity against a pathogen in an uninfected subjectcomprising contacting ex vivo dendritic cells obtained from said subjectwith an immunologically active lipopeptide of the invention or aderivative or variant thereof or a vaccine composition comprising saidlipopeptide or derivative or variant for a time and under conditionssufficient to confer epitope specific activity on T cells.

Accordingly, this aspect of the invention provides for theadministration of a prophylactic vaccine to the subject, wherein theactive substituent of said vaccine (i.e. the lipopeptide of theinvention) induces immunological memory via memory T cells in anuninfected individual. The preferred embodiments of vaccinationprotocols described herein for enhancing the cell mediated immunity of asubject apply mutatis mutandis to the induction of immunological memoryagainst the pathogen in a subject.

The present invention is further described with reference to thefollowing non-limiting examples and the drawings. The examples providedherein in mice are accepted models for equivalent diseases in humans andthe skilled person will readily be capable of extending the findingspresented herein for such models to a human disease context withoutundue experimentation.

EXAMPLE 1 Materials and Methods

Chemicals

Unless otherwise stated chemicals were of analytical grade or itsequivalent. N,N′-dimethylformamide (DMF), piperidine, trifluoroaceticacid (TFA), O′benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) anddiisopropylethylamine (DIPEA) and diisopropylcarbodiimide (DIPCDI) wereobtained from Auspep Pty. Ltd., Melbourne, Australia and Sigma-AldrichPty. Ltd., Castle Hill, Australia.O′benzotriazole-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU)was obtained from Bachem, (Bachem AG, Switzerland). Dichloromethane(DCM) and diethylether were from Merck Pty Ltd. (Kilsyth, Australia).Phenol and triisopropylsilane (TIPS) were from Aldrich (Milwaukee, Wis.)and trinitrobenzylsulphonic acid (TNBSA) and diaminopyridine (DMAP) fromFluka; 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was obtained from Sigmaand palmitic acid was from Fluka.

Viruses

The type A influenza viruses used in this study were an H3N1 subtypevirus referred to as Mem 71, which was derived by genetic reassortmentof A/Memphis/I/71 (H3N2) X A/Bellamy/42 (H1N1). Virus was grown for 2days in the allantoic cavity of 10-day embryonated hen's eggs. Allantoicfluid containing virus was stored in aliquots at −70° C. Infectiousvirus titers were obtained by assay of plaque formation in monolayers ofMadin-Darby canine kidney (MDCK) cells (Tannock et al, Infect. Immun.43, 457-462, 1984) and are expressed as PFU/milliliter.

Bacteria

Listeria monocytogenes EGD was cultured overnight at 37° C. on HorseBlood Agar (HBA) plates. The bacteria were washed off the plates usingsterile PBS and the concentration adjusted to 5×10³ Listeria cells/ml.Balb/c mice were infected intravenously with 1×10³ Listeria cells. Thedose was checked retrospectively by plating serial 10-fold dilutions onHBA plates.

Peptide Syntheses

Peptides Comprising Influenza Virus CTL Epitopes

A panel of immunogens was synthesized that incorporated peptidesrepresenting a minimal determinant for CD8+ T cells and/or a determinantfor CD4+ T cells, both from influenza virus. The peptide NP (147-155)with the sequence TYQRTRALV (a CTL determinant present in the NP of PR8virus; SEQ ID NO: 2) is the dominant CD8+ T-cell determinant recognizedby BALB/c mice and is common to all type A influenza virus strains(Bodmer et al, Cell 52, 253-258; 1988; and Sherman et al, J. Exp. Med.175, 1221-1226, 1992). The peptide HA2 (166-180), with the sequenceALNNRFQIKGVELKS (SEQ ID NO: 1), is a CD4⁺ T-helper determinant presentwithin the HA2 chain of Mem71 influenza virus hemagglutinin elicits CD4+T cells that are crossreactive with all viruses of the H3 subtype(Jackson et al, Virology 198, 153-170, 1994).

Peptides Comprising L. monocytocenes CTL Epitopes

An immunogenic peptide was synthesized that incorporated a minimal CTLepitope with amino acid sequence GYKDGNEYI (residues 91-99 of theprotein literialysin) from L. monocytogenes (ie. SEQ ID NO: 172) and aT-helper epitope from CDV-F (SEQ ID NO: 20).

Peptides comprising a CTL epitope expressed by B16-OVA tumour cell line.

An immunogenic peptide was synthesized that incorporated a CTL epitopewith amino acid sequence SIINFEKL (SEQ ID NO: 173) and a T-helperepitope from CDV-F (SEQ ID NO: 20).

Peptides comprising a CTL epitope from the core protein of hepatitis Cvirus.

An immunogenic peptide was synthesized that incorporated a CTL epitopewith amino acid sequence DLMGYIPLV (SEQ ID NO: 176) and a T-helperepitope from CDV-F (SEQ ID NO: 20).

General Procedures

Synthetic immunogens were assembled by conventional solid-phasemethodology using Fmoc chemistry. The general procedure used for thepeptide synthesis has been described by Jackson et al., Vaccine 18, 355(1999). To enable lipid attachment between the CD4⁺ T helper epitope andthe CTL epitope, Fmoc-lysine(Mtt)-OH was inserted at a point between thetwo epitopes in the approximate centre of the resin-bound peptide.Following completion of peptide synthesis the Mtt group was removed bycontinual flow washing with 1% TFA in dichloromethane over a period of30-45 mins.

Synthesis of Lipid Moieties of Formulae (I)

Pam₃Cys was prepared according to the method described by Weismuller etal., Hoppe Seylers Z Physiol Chem 364, 593 (1983), as modified accordingto the method described by Zeng et al., J Pept Sci 2, :66 (1996). Thelipoamino acid Pam₃Cys is coupled to the exposed epsilon-amino group oflysine according to the procedure described by Zeng et al. (supra).Briefly, a 2-fold excess of Pam₃Cys, TBTU and HOBt was dissolved in DCMand a 3-fold excess of DIPEA added. This solution was then added to theresin-bound peptide to generate the lipopeptide.

Synthesis of Lipid Moieties of Formulae (II)

The synthesis of Pam₂Cys was adapted from previously described methodsas described by Jones et al., Xenobiotica 5, 155 (1975) and Metzger etal., Int J Pept Protein Res 38, 545 (1991), with the exception that3-bromo-propan-1,2-diol was used instead of 3-chloro-propan-1,2-diol,and centrifugation and not filtration was used to recover the product.

Synthesis of Lipopeptides

Lipopeptides produced in this study had the general structures shown inFIG. 1. Amino acid sequences of the peptide moieties included in thevarious lipopeptides are shown in FIG. 2. Pam₂Cys was coupled topeptides according to the methods described by Jones et al., Xenobiotica5, 155 (1975.) and Metzger et al., Int J Pept Protein Res 38, 545(1991), with the following modifications:

I. Synthesis of S-(2,3-Dihydroxypropyl)cysteine

Triethylamine (6 g, 8.2 ml, 58 mmoles) was added to L-cysteinehydrochloride (3 g, 19 mmole) and 3-bromo-propan-1,2-diol (4.2 g, 2.36ml, 27 mmole) in water and the homogeneous solution kept at roomtemperature for 3 days. The solution was reduced in vacuo at 40° C. to awhite residue which was boiled with methanol (100 ml), centrifuged andthe residue dissolved in water (5 ml). This aqueous solution was addedto acetone (300 ml) and the precipitate isolated by centrifugation. Theprecipitate was purified by several precipitations from water withacetone to give S-(2,3-dihydroxypropyl)cysteine as a white amorphouspowder (2.4 g, 12.3 mmol, 64.7%).

II. Synthesis ofN-Fluorenylmethoxycarbonyl-S-(2,3-dihydroxypropyl)-cysteine(Fmoc-Dhc-OH)

S-(2,3-dihydroxypropyl)cysteine (2.45 g, 12.6 mmole) was dissolved in 9%sodium carbonate (20 ml). A solution offluorenylmethoxycarbonyl-N-hydroxysuccinimide (3.45 g, 10.5 mmole) inacetonitrile (20 ml) was added and the mixture stirred for 2 h, thendiluted with water (240 ml), and extracted with diethyl ether (25 ml×3).The aqueous phase was acidified to pH 2 with concentrated hydrochloricacid and was then extracted with ethyl acetate (70 ml×3). The extractwas washed with water (50 ml×2) and saturated sodium chloride solution(50 ml×2), dried over sodium sulfate and evaporated to dryness.Recrystallisation from ether and ethyl acetate at −20° C. yielded acolourless powder (2.8 g, 6.7 mmole, 63.8%).

III. Coupling of Fmoc-Dhc-OH to Resin-Bound Peptide

Fmoc-Dhc-OH (100 mg, 0.24 mmole) was activated in DCM and DMF (1:1, v/v,3 ml) with HOBt (36 mg, 0.24 mmole) and DICI (37 ul, 0.24 mmol) at 0° C.for 5 min. The mixture was then added to a vessel containing theresin-bound peptide (0.04 mmole, 0.25 g amino-peptide resin). Aftershaking for 2 h the solution was removed by filtration and the resin waswashed with DCM and DMF (3×30 ml each). The reaction was monitored forcompletion using the TNBSA test. If necessary a double coupling wasperformed.

IV. Palmitoylation of the Two Hydroxy Groups of the Fmoc-Dhc-PeptideResin

Palmitic acid (204 mg, 0.8 mmole), DICI (154 ul, 1 mmole) and DMAP (9.76mg, 0.08 mmole) were dissolved in 2 ml of DCM and 1 ml of DMF. Theresin-bound Fmoc-Dhc-peptide resin (0.04 mmole, 0.25 g) was suspended inthis solution and shaken for 16 h at room temperature. The solution wasremoved by filtration and the resin was then washed with DCM and DMFthoroughly to remove any residue of urea. The removal of the Fmoc groupwas accomplished with 2.5% DBU (2×5 mins).

All resin-bound peptide constructs were cleaved from the solid phasesupport with reagent B (88% TFA, 5% phenol, 2% TIPS, 5% water) for 2 hr,and purified by reversed phase chromatography as described by Zeng etal., Vaccine 18, 1031 (2000).

Analytical reversed phase high pressure liquid chromatography (RP-HPLC)was carried out using a Vydac C4 column (4.6×300 mm) installed in aWaters HPLC system and developed at a flow rate of 1 ml/min using 0.1%TFA in H₂O and 0.1% TFA in CH₃CN as the limit solvent. All productspresented as a single major peak on analytical RP-HPLC and had theexpected mass when analysed by MALDI-TOF mass spectrometry on a BrukerBIFLEX instrument equipped with delayed ion extraction.

In some cases two serine residues (Ser-Ser) were added between thepeptide and lipid moiety in which case serine residues were added to theε-amino group of the central lysine residue before the lipid moiety wasattached.

A schematic diagram of the peptides and lipopeptides used in this studyis shown in FIG. 1.

Immunization Protocols

Peptides Comprising Influenza Virus CTL Epitopes

Groups of female BALB/c mice, 6 to 8 weeks old, were inoculated at day 0and again on day 28. For subcutaneous (s.c.) inoculations 9 nmoles oflipopeptide constructs were prepared in 100 μl volume of saline per doseand non-lipidated peptides formulated as an emulsion in an equal volumeof complete Freund's adjuvant (CFA) for the primary injection orincomplete Freund's adjuvant for the secondary inoculation. Forintranasal (i.n.) inoculations, 9 nmoles of peptide in 50 μl of salinewere applied to the nares of mice anaesthetised with penthrane forinhalation.

Peptides Comprising a CTL Epitope of L. monocytogenes

5 BALB/c mice were inoculated with 9 nmoles of non-lipidated peptide([P25]-Lys-[LL091-99]), or lipidated peptide([P25]-Lys(Pam₂Cys-Ser-Ser)-[LL091-99]) in which lipid was attachedbetween the two epitopes at the approximate centre of the molecule, orwith 1000 bacteria: In the case of peptide vaccine, inoculation wassubcutaneous and in the case of bacteria inoculation was intravenous.The number of interferon-γ producing cells present in spleen wasmeasured on day 28 following in vitro stimulation with the CTL epitopeor no antigen. The vertical axis shows the number of interferon-γproducing cells per 1,000,000 splenocytes.

Peptides Comprising a CTL Epitope of Ovalbumin

Each of 9 C57BL/6 mice (8-10 wks) were immunised subcutaneously withnmoles of lipidated [P25]-Lys(Pam₂Cys-Ser-Ser)-[SIINFEKL] (SIINFEKLdisclosed as SEQ ID NO: 173) or non-lipidated [P25]-Lys-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO: 173) peptide in 100 μl volume ofsaline. In the case of lipidated peptide, lipid was attached between thetwo epitopes at the approximate centre of the molecule.

Peptides Comprising a CTL Epitope of Hepatitis C Virus Core Protein

Human monocyte-derived dendritic cells were incubated with lipopeptide[P25]-Lys(Pam₂Cys-Ser-Ser)-[HCV] (5 μg/mL) for 48 hours before stainingwith FITC-conjugated antibodies for HLA-DR, CD83 and CD86 beforeanalysis by flow cytometry.

Challenge of Immunized Mice with Influenza Virus

Penthrane anesthetized mice previously immunized with peptidescomprising CTL epitopes of influenza virus were challenged intranasally(i.n.) with 10^(4.5) PFU of infectious Mem 71 influenza virus. Eachmouse received 50 μl of virus in the form of allantoic fluid diluted inPBS. At 5 days after challenge; the mice were killed by cervicaldislocation, and the lungs were removed and transferred aseptically tobottles containing 1.5 ml of Hank's balanced salt solution supplementedwith 100 U of penicillin, 100 μg of streptomycin, and 30 μg ofgentamicin per ml. Lung homogenates were prepared by using a tissuehomogenizer, and the cell material was pelleted by centrifugation at300×g for 5 min. The supernatants were removed, divided into aliquotsand stored at −70° C. until required. Titers of infectious virus in thelung supernatants were determined by plaque assay on monolayers of MDCKcells (Tannock et al, Infect. Immun. 431, 457-462, 1984).

Challenge of Immunized Mice with L. monocytogenes

Mice immunized s.c. with 9 nmol peptide immunogen or PBS, or i.v. with1000 bacteria, were challenged by i.v. injection with bacteria 28 daysafter priming and the number of colony forming units of bacteria presentin the liver determined 28 days after challenge.

Challenge of Immunised Mice with Tumour Cells.

Melanoma Challenge.

14 days after inoculation with non-lipidated (P25]-Lys-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO: 173) or lipidated peptide[P25]-Lys(Pam₂Cys-Ser-Ser)-(SIINFEKL] (SIINFEKL disclosed as SEQ ID NO:173), 6 mice from each group were challenged with 2×10⁵ melanoma cellsexpressing ovalbumin [B16-OVA] and therefore expressed the CTL epitopeSIINFEKL (SEQ ID NO: 173) (Bellone, at al, J. Immunol. 165:2651-2656).Hair around the injection site was removed with an electric shaver priorto injection to facilitate measurement of the emerging tumors. Growingtumors were monitored, and the animals were sacrificed when tumor sizereached 15 by 15 mm. Mean tumor area was calculated for each treatmentgroup at the indicated number of days after the tumor challenge.

Lewis Lung Carcinoma Challenge.

Mice were injected with 3×10⁴ Lewis Lung tumour cells that had beentransfected with ovalbumin and therefore expressed the CTL epitope(Nelson et al., J. Immunol. 166: 5557-5566, 2001). Four days afterreceiving tumour cells, animals were inoculated with 20 nmoles oflipidated peptide [P25]-Lys(Pam₂Cys-Ser-Ser)-[SIINFEKL] (SIINFEKLdisclosed as SEQ ID NO: 173), non-lipidated peptide [P25]-Lys-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO: 173) or with PBS subcutaneously in thebase of the tail. A second dose of immunogen was administered elevendays after receiving the tumour cells. Animals were monitored for tumourincidence and survival; animals were euthanased when tumour areaexceeded 100 mm².

Tetramer Staining of Peptide-Specific CD8+ T Cells

CD8⁺ T cells specific for an immunodominant H-2K^(d)-restricted CTLepitope consisting of amino acid residues 147-155 of the nucleoproteinof influenza virus strain A/Puerto Rico/8/34 (PR8; H1N1) in thelipopeptide immunogen, as set forth in SEQ ID NO: 2, were identifiedusing tetrameric complexes of the H-2K^(d) glycoprotein with bound CTLpeptide (TYQRTRALV; SEQ ID NO: 2) (Bodmer et al, Cell 52: 253-258, 1988;Sherman et al, J. Exp. Med. 175: 1221-1226, 1992).

The monomer was a gift from Professor Peter Doherty, Department ofMicrobiology and Immunology, University of Melbourne and was made at St.Jude Children's Research Hospital, Memphis Tenn., USA. Tetramer was madeby incubating the monomer with Streptavidin-phycoerythrin (MolecularProbes, Eugene, Oreg., USA) at a 4:1 molar ratio.

Lymphocytes from the lung were first treated with 20 μL of normal mouseserum (NMS) for 5 mins at room temperature and then stained for 60 minwith the tetrameric complexes at a 1:25 dilution. This was followed bystaining with anti-CD8α, (53-6.7) conjugated with Allophycocyanin for 30mins on ice and washed twice and analysed by a fluorescence-activatedcell sorter (FACSort, Becton Dickinson, San Jose's, USA). The data wereanalysed by FlowJo (Tree Star, Inc, Calif., USA).

T-Cell Culture Medium

T-cell culture medium consisted of RPM1 1640 (CSL Ltd.) supplementedwith 10% (vol/vol) heat-inactivated fetal calf serum, 2 mM L-glutamine,2 mM sodium pyruvate, 30 μg of gentamicin/ml, 100 μg of streptomycin/ml,100 IU of penicillin/ml, and 10⁻⁴ M 2-mercaptoethanol.

Cytotoxic T-Cell Assays

Secondary effector cells were generated either from inguinal andpopliteal lymph nodes of mice that had been immunized s.c. 7 dayspreviously with lipopeptide immunogens or from spleen cells of miceprimed at least 28 days previously with the lipopeptide immunogens.Briefly, 4×10⁷ lymph node cells or spleen cells, depleted oferythrocytes by treatment with Tris-buffered ammonium chloride (0.15 MNH⁴Cl in 17 mM Tris-HCI at pH 7.2), were cultured with 10⁷ irradiated(2,200 rads, ⁶⁰Co source) virus-infected or lipopeptide-pulsed syngeneicspleen cells in 25-cm² tissue culture flasks (Falcon) containing 15 mlof T-cell culture medium. The virus-infected spleen cells had beenpreincubated at 37° C. for 30 min with 3,000 hemagglutinating units ofeither infectious Mem 71 or PR8 virus in 1 ml of serum-free RPM1 andwashed once prior to addition to the flask. The lipopeptide-pulsedspleen cells had been preincubated at 37° C. for 60 min with 100 μg ofthe CTL lipopeptide/ml and also washed once prior to addition to theflask. After 5 days of culture at 37° C. in a humidified atmospherecontaining 5% CO₂, the cells were washed three times and. used in⁵¹Cr-release assays. The ⁵¹Cr-release assays were performed intriplicate as described previously (Harling-McNabb et al, Int. Immunol.11, 1431-1439, 1999) by using P815 mastocytoma cells (H-2^(d), DBA/2) astargets.

In Vivo Cytotoxic T.-Cell Assays

The ability of various peptide-based immunogens to induceepitope-specific CTL was determined in vivo. Groups of three mice wereinoculated intranasally with various lipopeptides in 50 μl PBS andchallenged with Mem71 on day 28. In order to analyze CTL determinantspecific cytotoxicity in vivo, syngeneic spleen cells were pulsed withthe CTL determinant and labelled with high intensity CFSE (2.5 μM).Antigen-specific lysis was controlled by co-injecting syngeneic spleencells labelled with low intensity CFSE (0.25 μM). A mixture of 15×10⁶cells of each target cell population was injected intravenously on day 4post-infection. The mice were killed 16 hr later and spleens wereanalysed for the presence of CFSE-high and CFSE-low cell populations byflow cytometry. A total of 1×10⁶ lymphocytes were analysed for eachsample. Individual mice are represented by the closed squares and thebars represent the geometric mean titre.

ELISPOT Assay for IFN-γ-Secreting Cells

CTL-specific IFN-γ-secreting cells were enumerated by an ELISPOT assaymodified from that of Murali-Krishna et al, Immunity 8, 177-187, 1998.Flat-bottom polyvinyl chloride microtiter plates (96-well: Dynatech)were coated overnight with 50 μl of rat anti-(mouse IFN-γ) antibody(clone R4-14a2) at 5 μg/ml in PBS. Unoccupied sites on the wells werethen blocked by incubation for 1 h with 10 mg of bovine serum albumin/mlin PBS; and the plates were washed three times with PBS containing 0.05%Tween 20 (PBST). Twofold dilutions of spleen or lymph node cells inT-cell medium were then added to the wells, together with 5×10⁵irradiated (2,200 rads, ⁶⁰Co source) syngeneic spleen cells fromunimmunized mice and 10 U of recombinant human interleukin-2(Pharmingen, San Diego, Calif.)/well. Cells were incubated at 37° C. in5% CO₂ for 18 h the presence or absence of the CTL peptide at aconcentration of 1 μg of peptide/ml. Cells were then lysed and removedby rinsing the plate, initially with distilled water and then PBST.Then, 50 μl of a 1/500 dilution of biotinylated anti-(mouse IFN-γ)antibody (clone XMG 1.2; Pharmingen) was added, and the plates wereincubated at room temperature for 2 h. Plates were again washed, and 50μl of streptavidin-alkaline phosphatase (Pharmingen; 1/400 dilution in 5mg of bovine serum albumin/ml of PBST) was added to each well; themixtures were then incubated for a further 2 h. The plates were washed.and 100 μl of ELISPOT substrate (Sedgwick et al, J. Immunol. Methods 57,301-309, 1983) containing 1 mg of BCIP(5-bromo-4-chloro-3-indolylphosphate) per ml of2-amino-2-methyl-1-propynol buffer (Sigma) was added to each well. Whenblue-green spots had developed, the plates were washed with water anddried, and the spots were counted with the aid of an invertedmicroscope.

DI Dendritic Cell Cultures

Dendritic cells (DC) were cultured in medium based on complete IDDM.This consisted of Iscove's Modified Dulbecco's Medium (IMDM) containing25 mM HEPES and without alpha-thioglycerol or L-glutamine (JRHBioscience, Lenexa, USA), supplemented with 10° A) (v/v) heatinactivated (56° C., 30 min) foetal calf serum (CSL Ltd., Parkville,Victoria, Australia), gentamicin (24 μg/mL), glutamine (2 mM), sodiumpyruvate (2 mM), penicillin (100 IU/mL), streptomycin (180 μg/mL) and2-mercaptoethanol (0.1 mM). For DC generation complete IMDM was furthersupplemented with 30% supernatant from cultured NIH/3T3 cells and 5%GM-CSF in the form of a supernatant from Ag8653 cells transfected withthe GM-CSF gene (DC medium).

The culture method for immature dendritic cells was adapted from Winzleret al., J. Exp Med. 185, 317 (1997). Spleen cells from a BALB/c mousewere seeded at 1.5×10⁶ cells per 55 mm dish (Techno-Plas, S.A.,Australia) in 3 ml DC medium and incubated at 37° C. with 5% CO₂. Allthe equipment used for culturing was pyrogen free. The medium waschanged every 4 days and all cells returned to the dish. On day 12, bothsuspended and weakly adherent cells were collected by forcefullypipetting and then aspirating the medium. The procedure was repeatedwith 2 ml of PBS. The remaining strongly adherent cells were discarded.The collected cells were pelleted by centrifugation and reseeded into anew dish. Cells were subsequently maintained on a 4 day alternatingcycle of media change and passage. After 1 month of continuousculturing, the floating and semi-adherent cells took on the appearanceand staining characteristics of immature DC and are referred to as D1cells. Under these passage conditions the majority of cultured D1 cellsmaintain an immature phenotype characterized by an intermediateexpression level of cell surface MHC class II molecules.

Flow Cytometric Analysis of D1 Cells

D1 cells (1×10⁵ cells per sample) were seeded in a new Petri dish with 1mL of DC media and incubated with 0.0045 nmole of lipopeptide, dissolvedin complete IMDM medium. Lipopolysaccharide (LPS) purified from E. coliserotype 0111:B4 (Difco, Detroit, Mich., USA, was used at 5 μg/mL as apositive control for DC maturation. After overnight incubation, thecells were harvested and washed once with PBS with 1% FCS. To preventnon-specific binding to FCγRII/III, the cells were pre-incubated with 20μL of normal mouse serum for 5 mins at room temperature. The cells werethen exposed to FITC-conjugated monoclonal antibody 14-4-4S (IgG_(2a),anti-1-E^(k,d); Ozato et al., J. Immunol., 124, 533 (1980)) for 30 minon ice. Monoclonal antibody 36/1 (Brown et al., Arch Virol 114: 1,1990), which is specific for the antigen of influenza virus from whichthe T-helper epitope is derived, was used as an isotype control. Allantibodies were used at 2.5 μg/mL. The samples were washed once with PBScontaining 1% FCS and fixed with PBS containing 4% paraformaldehyde onice for 15 minutes. Flow cytometry analysis was performed using aFACSort (Becton Dickinson, San Jose, USA) and the data were analysedusing FlowJo software (Tree Star, Inc., San Carlos, Calif., USA).

Human Dendritic Cell Cultures

Generation of Monocyte-Derived Dendritic Cells

Peripheral blood mononuclear cells (PMBCs) were prepared from buffy coatpreparations obtained from blood donors (Red Cross Blood Bank,Melbourne, Australia) by Ficoll Paque (Amersham Pharmacia, Sweden)gradient separation. The cells were washed three-times in PBS andincubated with optimal amounts of murine anti-CD14 hybridoma supernatant(3C10, American Type Culture Collection) for 45 minutes on ice. Aftertwo washes, cells were further incubated with goat anti-murine IgGmicrobeads (Miltenyi Biotech, Germany) according to the manufacturer'sprotocol. CD14⁺ monocytes were then positively selected by affinitypurification using a magnet-activated cell sorting (MACS) column.Immature DC were generated by culturing the monocytes in GM-CSF and IL-4(40 ng/ml and 20 ng/ml, respectively [Schering Plough, USA])supplemented RPMI-1640 (Gibco, USA) containing 10% FCS(CSL, Australia),2 mmol/L glutamine, 2 mmol/L sodium pyruvate, 100 U/ml penicillin, 100μg/ml streptomycin, 30 μg/ml gentamicin and 0.1 mmol/L2-mercaptoethanol. Cells were cultured for 5 days before use with halfvolume changes of media every 2 days.

Measurement of DC Maturation

The ability of peptide and lipopeptide-based immunogens to up-regulatethe expression of MHC class II, CD83 and CD86 on human monocyte-deriveddendritic cells was determined by incubating 5×10⁵ cells per ml for 2days in medium supplemented with GM-CSF and IL-4 and either LPS (5μg/mL), non-lipidated peptide [Th]-Lys-[CTL] (5 μg/mL) or lipopeptide[Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] (5 μg/mL) for 48 hours. Phenotypicanalysis of surface markers was performed by staining withfluorochrome-conjugated monoclonal antibodies to HLA-DR (G46-6 [L243]),CD83 (HB15e), CD86 (Cat. No. 2331 [FUN-1]) and appropriate isotypematched antibodies (MOPC-21 and G155-178) from Becton Dickinson (USA),according to the manufacturer's protocols. Cells were then washed, fixedin 1% formaldehyde and analysed on a flow cytometer. The histograms arerepresentative of large granular cells gated on the forward and sidescatter dot plot. The shaded regions of the histograms and theassociated numerical values identify the percentage of cell populationsexpressing high levels of CD83, CD86 or HLA-DR.

EXAMPLE 2 Immunogenicity of Lipopeptides Comprising CTL Epitopes fromInfluenza Virus

Lipopeptides having a CTL epitope from influenza virus and in particularthe lipopeptides [Th]-Lys(Pam₂Cys-Ser-Ser)-(CTL) and[Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL], which comprise the amino acid sequenceset forth in SEQ ID NO: 4 were tested for their ability to induceenhanced CTL-mediated viral clearance and to enhance dendritic cellmaturation. As a negative control, a non-lipidated peptide having theamino acid sequence of SEQ ID NO: 4 was used in all experiments.

Viral Clearance

The lipopeptides elicited a higher level of viral clearance thannon-lipidated peptides (FIGS. 3, 4 a). Viral load in the lungs of miceprimed with the lipopeptides and challenged with infectious Mem 71 virus9 days later was reduced by 95% ([Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL]; FIG.3) or 99% ([Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL]; FIG. 3) compared to samplesfrom mice immunized with PBS alone. In contrast, non-lipidated peptideachieved only a 65% reduction in viral load ([Th]-Lys-(CTL); FIG. 3).Enhanced viral clearance was also observed in lipopeptide-inoculatedanimals that had been challenged with Mem 71 virus 28 days after theinitial inoculation. In contrast, the ability to clear virus issignificantly weaker at this time point in mice inoculated with thenon-lipidated peptide.

As shown in FIG. 4 b, there was also enhanced CD8⁺ T cell activation inimmunized mice receiving the lipopeptides referred to in the legend toFIG. 2, compared to mice receiving only non-lipidated peptide or PBS asseen by the number of CD8+ T cells found in the BAL fluids.

Dendritic Cell Maturation

The priming of naïve CD4+ T cells and CD8+ T cells in secondary lymphoidorgans by dendritic cells is preceded by maturation of DC upon exposureto antigen epitope. This maturation is characterised by up-regulation ofMHC products and co-stimulatory molecules on the DC surface. Wetherefore determined whether the various peptides and lipopeptides coulddifferentially activate dendritic cells in an attempt to explain thedifferent immunogenic properties of these vaccine candidates. Theresults of experiments in which a line of immature DC, D1 cells, wereexposed to peptides, stained for surface expression of MHC class IImolecules then analysed by flow cytometry, demonstrated that there wasenhanced maturation of dendritic cells following their exposure to thepeptides [Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL] or[Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] compared to [Th]-Lys-[CTL] peptide ormedium alone (FIG. 4 c).

[Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] was the most effective and thenon-lipidated peptide [Th]-Lys-[CTL] was the least effective in causingmaturation of DC, with [Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL] being nearly aseffective as [Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] (FIG. 4 c). The ability ofthe lipidated peptide [Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] to up-regulateclass II expression was the same as for bacterial lipopolysaccharide(LPS). The non-lipidated peptide was unable to induce maturation of D1cells greater than about 26%, a level that occurs spontaneously inculture. The relative abilities of these lipopeptides to inducematuration of D1 cells directly reflected their ability to induceCTL-mediated viral clearing responses and CD8+ T cells in the BAL.

Effects of Different Lipids on Cytotoxicity and T Cell Proliferation InVitro and In Vivo

The effects of conjugating different lipids, including Pam₁Cys, Pam₂Cys,Pam₃Cys, palmitic acid and cholesterol, to the peptide immunogen werealso determined:

As shown in FIG. 5, viral load in the lungs of mice primed withPam₂Cys-containing lipopeptides were lower than for mice primed withlipopeptides comprising the other lipids tested, suggesting that Pam₂Cysis preferred for conferring protection against virus. All lipidshowever, offered some protection against virus. This effect was alsoreflected in the IFN-gamma CD8+ T cell count (FIG. 6). Collectively,these data suggest that it is important to attach the lipid to thecysteine glycerol residue, as in the [Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL]epitope structure, for maximum cytotoxic effect.

In tetramer assays, the highest number of tetramer positive CD8+ T cellsper lung were observed for lipopeptides wherein the lipid moiety wasadded to the epsilon amino group of an internal lysine residue (e.g.,lipopeptides ITIA-Lys(PamiCysSer-Ser)-[CTL],[Th]-1-Ys(Pam2Cys-Ser-Ser)-[CTL], [TIA-Lys(Pam3Cys-Ser-Ser)-[CTL], and[Th]-Lys(Chol2Lys-Ser-Ser)-[CTL] in FIG. 7) compared to nonlipidatedpeptide or lipopeptide having lipid added to the N-terminus of thepeptide (e.g., construct Pal2LysLys[Th]-[CTL] in FIG. 7). These dataalso confirm that the positioning of the lipid internal to the peptide,by attachment to the epsilon amino group of an internal lysine residue,enhances cytotoxic activity of the CTL epitope.

To analyze CTL determinant specific cytotoxicity in vivo, mice wereinoculated intranasally with 9 nmoles of various lipopeptides in PBS andchallenged with Mem71 virus on day 28. CTL determinant-specificcytotoxicity in vivo was measured using syngeneic spleen cells pulsedwith the CTL determinant and labelled with high intensity CFSE.Non-pulsed spleen cells labelled with low intensity CFSE were used as acontrol. A mixture of cells of each target cell population was injectedintravenously on day 4 post-infection. The mice were killed 16 hr laterand spleens were analysed for the presence of CFSE-high and CFSE-lowcell populations by flow cytometry. A total of 1×10⁶ lymphocytes wereanalysed for each sample. Data in FIG. 8 is a graphical representationshowing cytotoxic T cell activity in naïve mice. FIG. 9 indicates thatthe lipopeptide [Th]-Lys(Pam₂Cys-Ser-Ser)-(CTL) comprising the CD4+T-helper epitope set forth in SEQ ID NO: 1 and the H-2^(d)-restrictedCIL epitope set forth in SEQ ID NO: 2, induced significant cytotoxicityin vivo.

As shown in FIG. 10, lipopeptides have higher activity thannon-lipidated peptide, with the lipopeptides designated[Th]-Lys(Pam₁Cys-Ser-Ser)-[CTL], [Th]-Lys(Pam₂Cys-Ser-Ser)-[CTL] and[Th]-Lys(Pam₃Cys-Ser-Ser)-[CTL] providing a marked enhancement ofspecific lysis in vivo compared to the non-lipidated peptide[Th]-Lys-(CTL) and other lipopeptides tested. These data again confirmthat positioning of the lipid internal to the peptide, by attachment tothe epsilon amino group of an internal lysine residue, enhancescytotoxic activity of the CTL epitope in vivo.

EXAMPLE 3 Immunogenicity of Lipopeptides Comprising a CTL Epitope fromL. monocytogenes

A lipopeptide having a CTL epitope from L. monocytogenes and inparticular the lipopeptide [P25]-Lys(Pam₂Cys-Ser-Ser)-[LL091-99]comprising the amino acid sequence set forth in SEQ ID NO: 175 wastested for its ability to induce a CD8+ T cell response, and to protectagainst a challenge with L. monocytogenes. As a negative control, PBS ora non-lipidated peptide having the amino acid sequence of SEQ ID NO: 175was used in all experiments. Isolated bacteria were used as a positivecontrol.

IFN-γ Production by Splenocytes

-   The lipopeptide tested in this study induced a specific CD8+ T cell    response against the immunizing CTL epitope, as evidenced by the    enhanced number of IFN-γ producing splenocytes present in mice    immunized with lipidated peptide relative to non-lipidated peptide.    Mice immunized with 9 nmoles lipidated peptide vaccine    [P25]-Lys(Pam₂Cys-Ser-Ser)-[LL091-99] comprising the amino acid    sequence set forth in SEQ ID NO: 175 produced about 15-fold more    IFN-γ producing cells per million splenocytes than mice receiving    non-lipidated peptide or a PBS control, indicating an enhanced    activation of IFN-γ producing CD8+ T cells in the mice. receiving    the lipidated peptide (FIG. 11).    Protection Against Challenge with Isolated Bacteria

Data in FIG. 12 indicate that the lipidated[P25]-Lys(Pam₂Cys-Ser-Ser)-[LL091-99] peptide successfully providesprotection against a subsequent challenge with whole bacteria. Asignificantly enhanced protection was also observed in mice immunizedwith the lipidated [P25]-Lys(Pam₂Cys-Ser-Ser)-(LL091-99] peptiderelative to mice immunized with non-lipidated [P25]-Lys-[LL091-99]peptide or PBS (i.e. non-immunized mice).

EXAMPLE 4 Protection Against Challenge with Tumour Cells

Protection Against Challenge with Melanoma Cells

The ability of the lipopeptide vaccine containing the ovalbumin CTLepitope (SIINFEKL) (SEQ ID NO: 173) to induce protection againstmelanoma cells expressing this CTL epitope (B16-OVA cells) was assessed.IFN-γ production was determined in mice inoculated with lipopeptidecomprising a CDV-F T-helper epitope (P25) and a CTL epitope (SIINFEKL)(SEQ ID NO: 173) of ovalbumin linked via the epsilon amino group of aninternal lysine residue positioned between said epitopes to Pam₂Cys(i.e. the peptide [P25]-Lys(Pam₂Cys-Ser-Ser)-[SIINFEKL](SIINFEKLdisclosed as SEQ ID NO: 173) listed in FIG. 2 and based upon SEQ ID NO:174). C57BL/6 mice were vaccinated with 20 nmoles lipidated peptide[P25]-Lys(Pam₂Cys-Ser-Ser)-[SIINFEKL] (SIINFEKL disclosed as SEQ ID NO:173), non-lipidated peptide [P25]-Lys-[SIINFEKL] (SIINFEKL disclosed asSEQ ID NO: 173) or with PBS subcutaneously in the base of the tail. Micewere then challenged subcutaneously on the back 14 days later withB16-OVA cells. Splenocytes were obtained from the inoculated animals andstimulated in vitro with the CTL epitope having the sequence SIINFEKL(SEQ ID NO: 173) and the number of IFN-γ producing cells per 1,000,000splenocytes was measured. Data show enhanced numbers of IFN-γ producingcells for mice inoculated with lipopeptide (Table 1), indicating anenhanced ability of the lipopeptides to activate T cells relative tonon-lipidated peptide.

Importantly, control of tumour growth was elicited by immunisation withlipopeptide compared to mice immunized with the non-lipidated peptide[P25)-Lys-[SIINFEKL] (SIINFEKL disclosed as SEQ ID NO: 173) or PBS alone(FIG. 13). No tumour growth was observed over a 15 day period in miceimmunised with [P25]-Lys(Pam₂Cys-Ser-Ser)SIINFEKL] (SIINFEKL disclosedas SEQ ID NO: 173). Conversely, tumours of greater than 75 mm² indiameter were observed in mice immunised with [P25]-Lys-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO: 173) or PBS alone. Together, thesedata confirm the protective ability of the lipopeptide compared tonon-lipidated peptide in protection against tumours.

TABLE 1 Numbers of IFN-γ secreting splenocytes inrepresentative melanoma samples receiving[P253-Lys(Pam₂Cys-Ser-Ser)-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO: 173)lipopeptide compared to non-lipidated [P25]-Lys-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO: 173) peptide or PBSPEPTIDE/LIPOPEPTIDE IMMUNOGEN [P25]-Lys(Pam₂Cys-Ser- [P25]-Lys-Ser)-[SIINFEKL [SIINFEKL] PBS No. IFN-γ secreting splenocytes per10⁶ splenocytes 284 18  5 205 14  0 192 10  0 Average 227 14  3Std. deviation  49  4 24Protection Against Challenge with Lewis Lung Tumour Cells

The ability of the lipopeptide to provide protection against Lewis Lungtumor development in animals in vivo was also tested. Mice were injectedwith 3×10⁴ Lewis Lung tumour cells transfected with ovalbumin andtherefore expressing the CTL epitope SIINFEKL (SEQ ID NO: 173) (Nelsonet al., J. Immunol. 166: 5557-5566, 2001). Four days after receivingtumour cells, animals were inoculated subcutaneously in the base of thetail with 20 nmoles lipidated peptide[P25]-Lys(Pam₂Cys-Ser-Ser)-[SIINFEKL] (SIINFEKL disclosed as SEQ ID NO:173), or alternatively, non-lipidated peptide [P25]-Lys-[SIINFEKL](SIINFEKL disclosed as SEQ ID NO: 173) or PBS. A second and similar doseof immunogen was administered eleven days after receiving the tumourcells. Data in FIG. 14 indicate that the percentage of animals withfewer lesions developing was significantly higher for animals receivingthe lipopeptide compared to animals receiving the non-lipidated peptideor PBS. As shown in FIG. 15, animals receiving the lipopeptide immunogenalso survived for longer than those receiving the non-lipidated peptideor PBS. These data further confirm the protective ability of thelipopeptide compared to non-lipidated peptide for protection againsttumours.

EXAMPLE 5 Enhanced Expression of MHC Class II, CD83 and CD86 on HumanDendritic Cells Following Administration of a Lipopeptide Comprising aCDV-F T-Helper Epitope and a CTL Epitope from Hepatitis C Virus

The lipopeptide [P251-Lys(Pam₂Cys-Ser-Ser)HCV] described in the legendto FIG. 2 was tested for its ability to up-regulate the expression ofMHC class II, CD83 and CD86 on human dendritic cells. Humanmonocyte-derived dendritic cells were incubated with media alone, LPS (5μg/mL), non-lipidated peptide [P25]-Lys-[HCV] (5 μg/mL) or lipopeptide[P25]-Lys(Pam₂Cys-Ser-Ser)-[HCV] (5 μg/mL) for 48 hours before stainingwith FITC-conjugated antibodies for HLA-DR, CD83 and CD86 beforeanalysis by flow cytometry. Data shown in FIG. 16 demonstrate a higherpercentage of dendritic cell populations that express HLA-DR, CD83 andCD86 antigens on their cell surface are present following treatment withlipidated peptide than following treatment with non-lipidated peptide orPBS alone. The ability of the lipopeptide to induce maturation of humandendritic cells directly reflected the immunogenic ability of thelipopeptide compared to the non-lipidated peptide, providing a possiblemechanism for immunogenicity.

EXAMPLE 6 Discussion

In this study we describe the assembly of a variety of lipopeptideimmunogens composed of a CD4⁺ T cell epitope, a CD8⁺ CTL epitope andPam₃Cys or Pam₂Cys linked thereto via the epsilon amino group of aninternal lysine residue.

The precise nature of the lipid moiety in generating an immune responsewas not shown to be critical, because a range of lipids, includingcholesterol, palmitic acid, Pam₁Cys, Pam₂Cys, and Pam₃Cys were shown tosuccessfully elicit T cell proliferation and cytotoxicity. However,significant differences were observed in terms of protection andIFN-gamma production, at least in the case of lipopeptides directedagainst influenza virus, suggesting that lipid structure may be animportant consideration in vivo. In particular, at least for vaccinesincorporating the influenza virus CTL epitope, Pam₂Cys linked to theepsilon amino group of an internal lysine residue in the peptide weremost effective in conferring protection, suggesting that a linkage tothe cysteine glycerol is preferred.

The lipopeptides of the invention are effective in enhancing the CD8+ Tcell responses of immunized animals against bacterial and viralpathogens and also against tumour cells. Given the success of theself-adjuvanting peptides exemplified herein to protect against viraland bacterial pathogens as well as tumour cells, it is reasonable toexpect that this technology is generally applicable to a wide range ofvaccination protocols.

Insertion of serine residues between the lipid moiety and the peptidesequence does not adversely affect the potency of the resultingPam₂Cys-containing immunogens.

The lipopeptides can trigger an immune response in the absence ofadditional adjuvant and can be delivered by both parenteral andnon-parenteral routes, particularly intranasally.

Taken together, the data provided herein demonstrate that placement of awide range of lipids, including but not limited to Pam₂Cys and Pam₃Cys,between the CTL epitope and the T helper epitope, at the approximatecentre of a totally synthetic peptide vaccine increases theimmunogenicity of the vaccine.

The invention claimed is:
 1. A lipopeptide comprising a polypeptideconjugated to one or more lipid moieties wherein: (i) said polypeptidecomprises an amino acid sequence wherein the linkages between the aminoacids making up the amino acid sequence consist of alpha-amino acidlinkages and the amino acid sequence comprises: (a) the amino acidsequence of a T helper cell (Th) epitope and the amino acid sequence ofa cytotoxic T cell (CTL) epitope, wherein said amino acid sequences aredifferent; and (b) one or more internal lysine residues or internallysine analog residues for covalent attachment of each of said lipidmoieties via the epsilon-amino group or terminal side-chain group ofsaid lysine or lysine analog; and (ii) each of said one or more lipidmoieties is covalently attached to an epsilon-amino group of said one ormore internal lysine residues or to a terminal side-chain group of saidone or more internal lysine analog residues, wherein an internal lysineresidue or internal lysine analog residue to which a lipid moiety isattached is positioned in the polypeptide amino acid sequence betweenthe Th epitope and the CTL epitope.
 2. The lipopeptide of claim 1wherein the lipid moiety has a structure of General Formula (VII):

wherein: (i) X is selected from the group consisting of sulfur, oxygen,disulfide (—S—S—), and methylene (—CH₂—), and amino (—NH—); (ii) m is aninteger being 1 or 2; (iii) n is an integer from 0 to 5; (iv) R₁ isselected from the group consisting of hydrogen, carbonyl (—CO—), andR′—CO— wherein R′ is selected from the group consisting of alkyl having7to 25 carbon atoms, alkenyl having 7 to 25 carbon atoms, and alkynylhaving 7 to 25 carbon atoms, wherein said alkyl, alkenyl or alkynylgroup is optionally substituted by a hydroxyl, amino, oxo, acyl, orcycloalkyl group; (v) R₂ is selected from the group consisting ofR′—CO—O—, R′—O—, R′—O—CO—, R′—NH—CO—, and R′—CO—NH—, wherein R′ isselected from the group consisting of alkyl having 7 to 25 carbon atoms,alkenyl having 7 to 25carbon atoms, and alkynyl having 7 to 25 carbonatoms, wherein said alkyl, alkenyl or alkynyl group is optionallysubstituted by a hydroxyl, amino, oxo, acyl, or cycloalkyl group; and(vi) R₃ is selected from the group consisting of R′—CO—O—, R′—O—,R′—O—CO—, R′—NH—CO—, and R′—CO—NH—, wherein R′ is selected from thegroup consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7to 25carbon atoms, and alkynyl having 7 to 25 carbon atoms, wherein saidalkyl, alkenyl or alkynyl group is optionally substituted by a hydroxyl,amino, oxo, acyl, or cycloalkyl group; and wherein each of R₁, R₂ and R₃are the same or different.
 3. The lipopeptide of claim 2 wherein X issulfur; m and n are both 1; R₁ is selected from the group consisting ofhydrogen, and R′—CO—, wherein R′ is an alkyl group having 7 to 25 carbonatoms; and R₂ and R₃ are selected from the group consisting of R′—CO—O—,R′—O—, R′—O—CO—, R′—NH—CO—, and R′—CO—NH—, wherein R′ is an alkyl grouphaving 7 to 25 carbon atoms.
 4. The lipopeptide of claim 3 wherein R′ isselected from the group consisting of: palmitoyl, myristoyl, stearoyl,lauroyl, octanoyl, decanoyl, and cholesterol.
 5. The lipopeptide ofclaim 2 wherein the lipid is contained within a lipoamino acid moietyselected from the group consisting of: Pam₁Cys, Pam₂Cys, Pam₃Cys,Chol₂Lys, Ste₂Cys, Lau₂Cys, and Oct₂Cys.
 6. The lipopeptide of claim 1wherein the lipid moiety has the following General Formula (VIII):

wherein: (i) R₄ is selected from the group consisting of: (i) analpha-acyl-fatty acid residue consisting of between about 7 and about 25carbon atoms; (ii) an alpha-alkyl-beta-hydroxy-fatty acid residue; (iii)a beta-hydroxy ester of an alpha-alkyl-beta-hydroxy-fatty acid residue;and (iv) a lipoamino acid residue; and (ii) R₅ is hydrogen or the sidechain of an amino acid residue.
 7. The lipopeptide of claim 1 whereinthe lipid moiety is separated from the peptide moiety by a spacer. 8.The lipopeptide of claim 7 wherein the spacer comprises arginine, serineor 6-aminohexanoic acid.
 9. The lipopeptide of claim 1 wherein theT-helper epitope is a T-helper epitope of influenza virus haemagglutininor a T-helper epitope of canine distemper virus F (CDV-F) protein. 10.The lipopeptide of claim 9 wherein the T-helper epitope of influenzavirus haemagglutinin comprises the amino acid sequence set forth in SEQID NO:
 1. 11. The lipopeptide of claim 9 wherein the T-helper epitope ofCDV-F protein comprises the amino acid sequence set forth in SEQ ID NO:20.
 12. The lipopeptide of claim 1 wherein the CTL epitope is from animmunogenic protein, lipoprotein, or glycoprotein of a virus.
 13. Thelipopeptide according to claim 12 wherein the virus is influenza virus.14. The lipopeptide according to claim 12 wherein the virus is hepatitisC virus.
 15. The lipopeptide of claim 1 wherein the CTL epitope is froman immunogenic protein, lipoprotein, or glycoprotein of a prokaryoticorganism.
 16. The lipopeptide of claim 1 wherein the CTL epitope is froman immunogenic protein, lipoprotein, or glycoprotein of a eukaryoticorganism.
 17. The lipopeptide according to claim 16 wherein the CTLepitope is from an ovalbumin protein of a mammal or a tumor cell.
 18. Acomposition comprising the lipopeptide of claim 1 and a pharmaceuticallyacceptable excipient or diluent.